**Abstract**

Neutrophil leukocytes provide first-line phagocytic defense against infection. Phagocyte locomotion to the site of infection, identification, and phagocytosis of the infecting microbe results in metabolically driven O2-dependent combustive microbicidal action. NADPH oxidase activity controls this respiratory burst metabolism. Its flavoenzyme character allows semiquinone-mediated crossover from two reducing equivalents (2RE) to 1RE transfer, as is necessary for univalent reduction of O2 to the acid hydroperoxyl radical (HO2) and its conjugate base, superoxide anion (O2 <sup>−</sup>). RE transfer dynamics is considered from the perspectives of quantum and particle physics, as well as frontier orbital interactions. Direct disproportionation of HO2-O2 <sup>−</sup> yields electronically excited singlet molecular oxygen (1 O2\*) and hydrogen peroxide (H2O2). Myeloperoxidase catalyzes H2O2-dependent 2RE oxidation of chloride (Cl<sup>−</sup>) to hypochlorite (OCl<sup>−</sup>). Direct nonenzymatic reaction of OCl<sup>−</sup> with an additional H2O2 yields Cl<sup>−</sup>, H2O, and <sup>1</sup> O2\*. Thus, for two 2RE metabolized through NADPH oxidase, a total of three <sup>1</sup> O2\* are possible. H2O2, OCl<sup>−</sup>, and <sup>1</sup> O2\* generated are all singlet multiplicity reactants and can participate in spin-allowed combustive oxygenations yielding light emission, that is, luminescence or chemiluminescence. The sensitivity of luminescence for measuring neutrophil redox activities is increased several orders of magnitude by introducing chemiluminigenic probes. Probes can be selected to differentiate oxidase from haloperoxidase activities.

**Keywords:** neutrophil, respiratory burst, reducing equivalent, combustion, frontier orbital, spin quantum number, NADPH oxidase, myeloperoxidase, Wigner spin conservation, Hund's maximum multiplicity rule, boson, fermion

## **1. Introduction**

*There is a complicated hypothesis, which usually entails an element of mystery and several unnecessary assumptions. This is opposed by a more simple explanation, which contains no unnecessary assumptions. The complicated one is always the popular one at first, but the simpler one, as a rule, eventually is found to be correct. This process* 

*frequently requires 10–20 years. The reason for this long time lag was explained by Max Planck. He remarked that scientists never change their minds, but eventually they die.*

John H. Northrop, 1961 [1].

**63**

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action…*

The neutrophil "respiratory burst" describes the large increases in glucose consumption via the hexose monophosphate shunt (aka, pentose pathway) [5, 6], and in nonmitochondrial molecular oxygen (O2) consumption [7] associated with phagocytosis, and required for microbicidal action. Appreciating the underlying necessity for such metabolic changes provides perception into oxygen chemistry and biochemistry, radical reactivity and combustion in general. The character of electron transfer mediated by the dehydrogenases of the hexose monophosphate (HMP) shunt is common to cytoplasmic redox reactions. Such oxidation-reduction transfers typically involve movement of two reducing equivalents (2RE), that

a dehydrogenase. In turn, the dehydrogenase mobilizes the 2RE by transfer to

form NAD(P)H. The cofactors NADPH and NADH serve as the cytoplasmic redox carriers for 2RE transfers between dehydrogenases and oxidases, and are common to various pathways of cytoplasmic metabolism. Consumption of 2RE carried

. Availability of NADP+

producing NADPH. The point for emphasis is that 2RE are transferred, not

Respiratory burst metabolism results from the activation of NADPH oxidase. Like many oxidases, NADPH oxidase is a flavoenzyme. Flavoenzymes are mechanistically unique in that 2RE reduction, by cofactors such as NAD(P)H, is followed by a series of 1RE oxidations. In its 1RE form, the riboflavin prosthetic group of flavin adenine dinucleotide (FAD) is in the semiquinone state [9, 10]. This semiquinone capability, usually in combination with a cytochrome component, allows the oxidase to transition from 2RE transfer to 1RE transfers. As such, flavoenzymes are the junction enzymes where 2RE transfer proceeds as 1RE cytochrome transfers, for example, the mitochondrial electron transport system or the microsomal cytochrome-P450 mixedfunction oxidase system [10, 11]. Flavoprotein oxidases are also capable of catalyzing the 1RE reduction of O2 [12, 13]. As such, phagocytosis-associated activation of NADPH oxidase opens the possibility for univalent, that is, 1RE, reduction of O2. The molecular oxygen we breathe has unique physical-chemical characteristics. In its ground, that is, lowest energy state, oxygen is a diradical, paramagnetic mol-

O2; the preceding superscripted (3

O2

shunt dehydrogenase activity. Dehydrogenation is a type of oxidation that does not require or directly involve O2. Glucose-6-phosphate (G-6-P) dehydrogenase, the initiator enzyme of the HMP shunt removes a total of 2RE and transfers the 2RE to

one. Such 2RE transfer, sometimes referred to as hydride ion (H<sup>−</sup>) transfer, is the

nicotinamide adenine dinucleotide (phosphate) NAD(P)<sup>+</sup>

), from an organic substrate catalyzed by

generating its reduced

is rate limiting for HMP

) indicates

HO2) and

O2 to participate

<sup>−</sup>) [2, 4, 14, 15]. Such reduction

*DOI: http://dx.doi.org/10.5772/intechopen.81543*

is, 2 electrons (e<sup>−</sup>) and 2 protons (H+

by NADPH returns it to NADP+

rule for cytoplasmic redox reactions [8].

ecule with triplet spin multiplicity [3

its conjugate base, the superoxide anion radical (2

multiplicity]. These spin characteristics guarantee a tendency for <sup>3</sup>

does not produce radical character; it decreases such character.

**2.1 Bosonic character of coupled fermionic electron transfer**

in 1RE reduction yielding the doublet multiplicity hydroperoxyl radical (2

Movement of 2RE is the transfer of an electron couple, that is, an orbital pair of electrons. Such 2RE transfers are the rule in cytoplasmic redox reactions. Considered from the perspective of particle physics, movement of a single electron (1RE) is quite different from paired electron (2RE) movement. Transfer of 1RE is a fermionic transfer. An electron is a fermion, and fermions have wave functions that are antisymmetric to exchange of particles; that is, Ψ (*a*, *b*) = −Ψ (*b*, *a*). Fermions anti-commute; that is, *a* × *b* ≠ *b* × *a*. Rotating a fermion through 360°,

NADP+

**2. Respiratory burst**

Appreciating why combustion is not spontaneous, how electrons are transferred biologically, and the unusual nature of oxygen reactivity were difficult for me as a student. So, in addition to biochemical studies, my mentor Richard Steele suggested I study the writings of Herzberg and others. Although challenging, such exposure shook open the door to other perspectives. Fundamental quantum and particle physics considerations were entertained with regard to oxygen and biologic electron transfer. My epiphany was in recognizing that the polymorphonuclear neutrophil, a leukocyte familiar to me from clinical laboratory experience, might realize the electronegative potential of oxygen for combustive microbicidal action by changing the spin multiplicity of oxygen. The following, taken from a symposium abstract presented in 1972, succinctly describes that position [2]. "Recently, a chemiluminescence (CL) has been observed when human polymorphonuclear leukocytes (PMN) phagocytize bacteria or particulate matter. The CL response correlates well with the stimulation of the hexose monophosphate shunt, which results in the generation of NADPH. The PMN possesses both CN<sup>−</sup>-insensitive NADH and NADPH oxidases. Flavoproteins oxidases of this type are capable of univalent reduction of O2. The reduced oxygen (·O2 <sup>−</sup>, ·O2H) may then disproportionate yielding HOOH and singlet molecular oxygen <sup>1</sup> O2. The PMN also possesses a CN<sup>−</sup>-sensitive peroxidase, myeloperoxidase, which has microbicidal activity in the presence of HOOH and halide. In this reaction, the HOOH is reduced to OH<sup>−</sup> with the oxidation of the halide to a reactive halogonium species. In cases where the halogonium formed is Cl<sup>+</sup> or Br<sup>+</sup> , there is potential for further reaction with HOOH resulting in the generation of a haloperoxy anion. This unstable species can disintegrate to yield the original halide and <sup>1</sup> O2. <sup>1</sup> O2 has been demonstrated to be a potent microbicidal agent. Therefore, the biochemical generation of <sup>1</sup> O2 by the PMN might be closely associated with microbicidal activity. The CL response may be the result of the relaxation of excited carbonyl groups generated via <sup>1</sup> O2-mediated oxidations."

Neutrophil leukocytes and monocytes play an essential role in innate phagocytic defense against infection. Immune surveillance mechanisms detect the presence of potentially pathologic microbes and generate the chemical signals that mobilize circulating neutrophils and prime the expression of receptors necessary for neutrophil navigation and phagocytosis. Contact of a primed neutrophil with activated endothelium is followed by neutrophil diapedesis into the tissue interstitial space, and locomotion to the site of infection guided by concentration gradients of complement anaphylatoxin, microbial products, cytokines, and lipid factors. Once an immunologically primed neutrophil contacts an opsonin-labeled pathogen, phagocytosis occurs. Phagocytosis is associated with a constellation of metabolic changes classically referred to as the "respiratory burst" [3]. This presentation focuses on the neutrophil redox mechanisms necessary for microbicidal action, especially the roles of NADPH oxidase and myeloperoxidase (MPO) in lethal microbicidal oxygenations. The Merriam-Webster dictionary defines combustion as a chemical reaction that occurs when oxygen combines with other substances to produce heat and usually light. By changing the spin multiplicity of oxygen from triplet to doublet, and then to singlet, neutrophils remove the spin barrier to direct oxygenation, enabling direct oxygen combustive microbicidal action with associated light emission, that is, chemiluminescence or luminescence [4].

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action… DOI: http://dx.doi.org/10.5772/intechopen.81543*

## **2. Respiratory burst**

*Neutrophils*

*frequently requires 10–20 years. The reason for this long time lag was explained by Max Planck. He remarked that scientists never change their minds, but eventually they die.*

Appreciating why combustion is not spontaneous, how electrons are transferred biologically, and the unusual nature of oxygen reactivity were difficult for me as a student. So, in addition to biochemical studies, my mentor Richard Steele suggested I study the writings of Herzberg and others. Although challenging, such exposure shook open the door to other perspectives. Fundamental quantum and particle physics considerations were entertained with regard to oxygen and biologic electron transfer. My epiphany was in recognizing that the polymorphonuclear neutrophil, a leukocyte familiar to me from clinical laboratory experience, might realize the electronegative potential of oxygen for combustive microbicidal action by changing the spin multiplicity of oxygen. The following, taken from a symposium abstract presented in 1972, succinctly describes that position [2]. "Recently, a chemiluminescence (CL) has been observed when human polymorphonuclear leukocytes (PMN) phagocytize bacteria or particulate matter. The CL response correlates well with the stimulation of the hexose monophosphate shunt, which results in the generation of NADPH. The PMN possesses both CN<sup>−</sup>-insensitive NADH and NADPH oxidases. Flavoproteins oxidases of this type are capable of

a CN<sup>−</sup>-sensitive peroxidase, myeloperoxidase, which has microbicidal activity in the presence of HOOH and halide. In this reaction, the HOOH is reduced to OH<sup>−</sup> with the oxidation of the halide to a reactive halogonium species. In cases where the

resulting in the generation of a haloperoxy anion. This unstable species can disinte-

Neutrophil leukocytes and monocytes play an essential role in innate phagocytic defense against infection. Immune surveillance mechanisms detect the presence of potentially pathologic microbes and generate the chemical signals that mobilize circulating neutrophils and prime the expression of receptors necessary for neutrophil navigation and phagocytosis. Contact of a primed neutrophil with activated endothelium is followed by neutrophil diapedesis into the tissue interstitial space, and locomotion to the site of infection guided by concentration gradients of complement anaphylatoxin, microbial products, cytokines, and lipid factors. Once an immunologically primed neutrophil contacts an opsonin-labeled pathogen, phagocytosis occurs. Phagocytosis is associated with a constellation of metabolic changes classically referred to as the "respiratory burst" [3]. This presentation focuses on the neutrophil redox mechanisms necessary for microbicidal action, especially the roles of NADPH oxidase and myeloperoxidase (MPO) in lethal microbicidal oxygenations. The Merriam-Webster dictionary defines combustion as a chemical reaction that occurs when oxygen combines with other substances to produce heat and usually light. By changing the spin multiplicity of oxygen from triplet to doublet, and then to singlet, neutrophils remove the spin barrier to direct oxygenation, enabling direct oxygen combustive microbicidal action with associated light emission, that is,

O2. <sup>1</sup>

might be closely associated with microbicidal activity. The CL response may be

univalent reduction of O2. The reduced oxygen (·O2

halogonium formed is Cl<sup>+</sup>

oxidations."

grate to yield the original halide and <sup>1</sup>

chemiluminescence or luminescence [4].

tionate yielding HOOH and singlet molecular oxygen <sup>1</sup>

or Br<sup>+</sup>

microbicidal agent. Therefore, the biochemical generation of <sup>1</sup>

the result of the relaxation of excited carbonyl groups generated via <sup>1</sup>

John H. Northrop, 1961 [1].

<sup>−</sup>, ·O2H) may then dispropor-

, there is potential for further reaction with HOOH

O2 has been demonstrated to be a potent

O2. The PMN also possesses

O2 by the PMN

O2-mediated

**62**

The neutrophil "respiratory burst" describes the large increases in glucose consumption via the hexose monophosphate shunt (aka, pentose pathway) [5, 6], and in nonmitochondrial molecular oxygen (O2) consumption [7] associated with phagocytosis, and required for microbicidal action. Appreciating the underlying necessity for such metabolic changes provides perception into oxygen chemistry and biochemistry, radical reactivity and combustion in general. The character of electron transfer mediated by the dehydrogenases of the hexose monophosphate (HMP) shunt is common to cytoplasmic redox reactions. Such oxidation-reduction transfers typically involve movement of two reducing equivalents (2RE), that is, 2 electrons (e<sup>−</sup>) and 2 protons (H+ ), from an organic substrate catalyzed by a dehydrogenase. In turn, the dehydrogenase mobilizes the 2RE by transfer to nicotinamide adenine dinucleotide (phosphate) NAD(P)+ generating its reduced form NAD(P)H. The cofactors NADPH and NADH serve as the cytoplasmic redox carriers for 2RE transfers between dehydrogenases and oxidases, and are common to various pathways of cytoplasmic metabolism. Consumption of 2RE carried by NADPH returns it to NADP+ . Availability of NADP+ is rate limiting for HMP shunt dehydrogenase activity. Dehydrogenation is a type of oxidation that does not require or directly involve O2. Glucose-6-phosphate (G-6-P) dehydrogenase, the initiator enzyme of the HMP shunt removes a total of 2RE and transfers the 2RE to NADP+ producing NADPH. The point for emphasis is that 2RE are transferred, not one. Such 2RE transfer, sometimes referred to as hydride ion (H<sup>−</sup>) transfer, is the rule for cytoplasmic redox reactions [8].

Respiratory burst metabolism results from the activation of NADPH oxidase. Like many oxidases, NADPH oxidase is a flavoenzyme. Flavoenzymes are mechanistically unique in that 2RE reduction, by cofactors such as NAD(P)H, is followed by a series of 1RE oxidations. In its 1RE form, the riboflavin prosthetic group of flavin adenine dinucleotide (FAD) is in the semiquinone state [9, 10]. This semiquinone capability, usually in combination with a cytochrome component, allows the oxidase to transition from 2RE transfer to 1RE transfers. As such, flavoenzymes are the junction enzymes where 2RE transfer proceeds as 1RE cytochrome transfers, for example, the mitochondrial electron transport system or the microsomal cytochrome-P450 mixedfunction oxidase system [10, 11]. Flavoprotein oxidases are also capable of catalyzing the 1RE reduction of O2 [12, 13]. As such, phagocytosis-associated activation of NADPH oxidase opens the possibility for univalent, that is, 1RE, reduction of O2.

The molecular oxygen we breathe has unique physical-chemical characteristics. In its ground, that is, lowest energy state, oxygen is a diradical, paramagnetic molecule with triplet spin multiplicity [3 O2; the preceding superscripted (3 ) indicates multiplicity]. These spin characteristics guarantee a tendency for <sup>3</sup> O2 to participate in 1RE reduction yielding the doublet multiplicity hydroperoxyl radical (2 HO2) and its conjugate base, the superoxide anion radical (2 O2 <sup>−</sup>) [2, 4, 14, 15]. Such reduction does not produce radical character; it decreases such character.

#### **2.1 Bosonic character of coupled fermionic electron transfer**

Movement of 2RE is the transfer of an electron couple, that is, an orbital pair of electrons. Such 2RE transfers are the rule in cytoplasmic redox reactions. Considered from the perspective of particle physics, movement of a single electron (1RE) is quite different from paired electron (2RE) movement. Transfer of 1RE is a fermionic transfer. An electron is a fermion, and fermions have wave functions that are antisymmetric to exchange of particles; that is, Ψ (*a*, *b*) = −Ψ (*b*, *a*). Fermions anti-commute; that is, *a* × *b* ≠ *b* × *a*. Rotating a fermion through 360°,

Ψ — 360° → −Ψ, changes the phase, but does not return the fermion to its original state. An additional 360° rotation, −Ψ —360° → Ψ, is required to return the antisymmetric particle to its original state [16]. Fermions obey Fermi-Dirac statistics.

A fermionic electron is defined by its five quantum numbers: *n*, *l*, *ml*, *s*, and *ms* [17]. The spin number, *s*, describes the intrinsic angular momentum of the electron independent of orbital motion, and has a value of ½*ħ* (abbreviated to ½). This quality has no analogy in classical physics. The total spin angular momentum, *S*, of an atom or molecule is expressed by the equation *S* = √[*s*(*s* + 1)]*ħ*. *s* gives rise to the quantum number *ms*, and only two values are allowed. When *ms* = ½, the *e −* is described as spin up (↑); when *ms* = −½, the *e −* is described as spin down (↓). The Pauli exclusion principle states that the total wave function for a system must be antisymmetric to the exchange of any pair of electrons. Differently stated, no two electrons of a given atom or molecule can have identical quantum numbers, and for two electrons to occupy an orbital, each electron must have opposite spins, that is, one orbital *e* <sup>−</sup> must have an *ms* = ½ (↑), the other orbital *e* <sup>−</sup> must have an *ms* = −½ (↓). Consequently, the total spin quantum number, *S*, for a filled orbital electron-couple is ½ + −½ = 0 (↑↓).

Bosons obey Bose-Einstein statistics, and have wave functions that are symmetric to exchange of a pair of particles; that is, Ψ (*a*, *b*) = Ψ (*b*, *a*). They obey ordinary commutation, that is, *a* × *b* = *b* × *a*. Rotating a boson through 360°, Ψ — 360° **→** *Ψ*, returns it to its original state. Bosons, for example, photons are symmetric particles with integral spin. Likewise, a spin-balanced composite of fermionic particles, for example, an alpha particle with an *S* of 0, is bosonic. With regard to biochemical redox reactions, the coupling of antisymmetric fermions, for example, the coupled electrons of an orbital pair, result in a *S* = 0 state with bosonic symmetry. The transfer of 2RE describes the movement of a coupled electron pair with an *S* = 0 and is in essence a bosonic transfer.

#### **2.2 Bosonic versus fermionic frontier orbital interactions**

Chemistry is about the frontier orbital interactions of atoms and molecules [18]. The focus of frontier orbital theory is on the initial orbital conditions of the reactants and on reactive transition to product(s) with emphasis on the highest occupied atomic or molecular orbital (HO(A)MO) and the lowest unoccupied atomic or molecular (LU(A)MO) orbital. The frontier orbital of a radical reactant is neither empty nor completely filled, and as such, is described as a singly occupied atomic or molecular orbital (SOAO or SOMO). Atomic and molecular orbitals, including frontier orbitals, can have bosonic or fermionic character [19, 20]. A HO(A)MO has an *S* = 0. Such an atom or molecule has singlet spin multiplicity with nonradical, diamagnetic character. A radical SO(A)MO has an *S* = ½ or −½, and has doublet spin multiplicity with radical, paramagnetic character.

The bosonic character of the HOMO of a nonradical reactant differs fundamentally from the fermionic character of the SOMO of a radical reactant. The fermionic nature of a SOMA limits overlap possibilities with bosonic HOMO. If such reaction occurs, the fermionic character must be preserved in the product. The electronegative Fenton radical (2 OH) can extract 1RE from the HOMO of a singlet multiplicity nonradical substrate (1 substrate) yielding singlet multiplicity 1 H2O, but in the process the HOMO of the substrate is converted to a SOMO, that is, the substrate becomes a doublet multiplicity radical (2 substrate). The symmetry of the reactants is preserved in the products. If a fermionic (doublet)-bosonic (singlet) reaction occurs, symmetry will be retained in the bosonic (singlet)-fermionic (doublet) products. Consistent with the Wigner-Witmer rules described in **Table 1**, spin symmetry is conserved [19–22].

The fermionic character of two radical reactants is eliminated in reactive bonding yielding a bosonic product. As described in **Table 1**, fermionic radical-radical, SOMO-SOMO reaction yields bosonic nonradical product. Simply stated, radicals

**65**

**Figure 1.**

*energy of 1*

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action…*

tend to react with radicals, and such doublet-doublet annihilations yield nonradical, that is, bosonic, product. Such reaction is responsible for terminating radical chain

Molecular oxygen in its ground state has unique triplet spin multiplicity [23]. Its two degenerate, that is, equal energy, frontier orbitals are each populated by a single electron. These two SOMO electrons obey Hund's maximum multiplicity rule, that is, the electron in each degenerate SOMO will have the same spin [24]. As illustrated in **Figure 1**, the *S* value for molecular oxygen is ½ + ½ or −½ + −½, and thus, the multiplicity is triplet, that is, 2|1 or −1| + 1 = 3. This bi-radical, bi-fermionic charac-

O2 predicts potential for highly exergonic reactions with nonradical, singlet multiplicity organic molecules, but thermodynamic potential does not guarantee reactivity, and combustion is not spontaneous. Taking a different perspective, it is

> Singlet bosonic

Doublet fermionic

Triplet bi-fermionic

Singlet bosonic

Doublet fermionic

Singlet bosonic

*Triplet and electronically excited singlet molecular oxygen with emphasis on the π\* (pi antibonding) frontier orbitals. The two π\* are degenerate (same energy level). Hund's maximum multiplicity rule predicts lowest* 

*O2.*

*energy is achieved when each SOMO electron has the same spin, that is, the triplet state (<sup>3</sup>*

*O2\* is 22.5 kcal/mol (94.2 kJ/mol) above <sup>3</sup>*

*Spin multiplicity states with regard to the bosonic-fermionic character of reactants and products.*

O2. The high electronegativity

*O2). The electronic* 

*DOI: http://dx.doi.org/10.5772/intechopen.81543*

ter is responsible for the paramagnetic character of 3

**Reactants Products**

propagation reactions.

Singlet + Singlet bosonic + bosonic

Singlet + Doublet bosonic + fermionic

Singlet + Triplet bosonic + bi-fermionic

Doublet + Doublet fermionic + fermionic

Doublet + Triplet fermionic + bi-fermionic

Triplet + Triplet

*Spin conservation rules.*

**Table 1.**

bi-fermionic + bi-fermionic

of 3

### *Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action… DOI: http://dx.doi.org/10.5772/intechopen.81543*

tend to react with radicals, and such doublet-doublet annihilations yield nonradical, that is, bosonic, product. Such reaction is responsible for terminating radical chain propagation reactions.

Molecular oxygen in its ground state has unique triplet spin multiplicity [23]. Its two degenerate, that is, equal energy, frontier orbitals are each populated by a single electron. These two SOMO electrons obey Hund's maximum multiplicity rule, that is, the electron in each degenerate SOMO will have the same spin [24]. As illustrated in **Figure 1**, the *S* value for molecular oxygen is ½ + ½ or −½ + −½, and thus, the multiplicity is triplet, that is, 2|1 or −1| + 1 = 3. This bi-radical, bi-fermionic character is responsible for the paramagnetic character of 3 O2. The high electronegativity of 3 O2 predicts potential for highly exergonic reactions with nonradical, singlet multiplicity organic molecules, but thermodynamic potential does not guarantee reactivity, and combustion is not spontaneous. Taking a different perspective, it is


## **Table 1.**

*Neutrophils*

Ψ — 360° → −Ψ, changes the phase, but does not return the fermion to its original state. An additional 360° rotation, −Ψ —360° → Ψ, is required to return the antisymmetric particle to its original state [16]. Fermions obey Fermi-Dirac statistics. A fermionic electron is defined by its five quantum numbers: *n*, *l*, *ml*, *s*, and *ms* [17]. The spin number, *s*, describes the intrinsic angular momentum of the electron independent of orbital motion, and has a value of ½*ħ* (abbreviated to ½). This quality has no analogy in classical physics. The total spin angular momentum, *S*, of an atom or molecule is expressed by the equation *S* = √[*s*(*s* + 1)]*ħ*. *s* gives rise to the quantum

principle states that the total wave function for a system must be antisymmetric to the exchange of any pair of electrons. Differently stated, no two electrons of a given atom or molecule can have identical quantum numbers, and for two electrons to occupy

Bosons obey Bose-Einstein statistics, and have wave functions that are symmetric to exchange of a pair of particles; that is, Ψ (*a*, *b*) = Ψ (*b*, *a*). They obey ordinary commutation, that is, *a* × *b* = *b* × *a*. Rotating a boson through 360°, Ψ — 360° **→** *Ψ*, returns it to its original state. Bosons, for example, photons are symmetric particles with integral spin. Likewise, a spin-balanced composite of fermionic particles, for example, an alpha particle with an *S* of 0, is bosonic. With regard to biochemical redox reactions, the coupling of antisymmetric fermions, for example, the coupled electrons of an orbital pair, result in a *S* = 0 state with bosonic symmetry. The transfer of 2RE describes the movement of a coupled electron pair with an *S* = 0 and is in essence a bosonic transfer.

Chemistry is about the frontier orbital interactions of atoms and molecules [18]. The focus of frontier orbital theory is on the initial orbital conditions of the reactants and on reactive transition to product(s) with emphasis on the highest occupied atomic or molecular orbital (HO(A)MO) and the lowest unoccupied atomic or molecular (LU(A)MO) orbital. The frontier orbital of a radical reactant is neither empty nor completely filled, and as such, is described as a singly occupied atomic or molecular orbital (SOAO or SOMO). Atomic and molecular orbitals, including frontier orbitals, can have bosonic or fermionic character [19, 20]. A HO(A)MO has an *S* = 0. Such an atom or molecule has singlet spin multiplicity with nonradical, diamagnetic character. A radical SO(A)MO has an *S* = ½ or −½, and has doublet

The bosonic character of the HOMO of a nonradical reactant differs fundamentally from the fermionic character of the SOMO of a radical reactant. The fermionic nature of a SOMA limits overlap possibilities with bosonic HOMO. If such reaction occurs, the fermionic character must be preserved in the product. The electronega-

substrate) yielding singlet multiplicity 1

the HOMO of the substrate is converted to a SOMO, that is, the substrate becomes a

in the products. If a fermionic (doublet)-bosonic (singlet) reaction occurs, symmetry will be retained in the bosonic (singlet)-fermionic (doublet) products. Consistent with the Wigner-Witmer rules described in **Table 1**, spin symmetry is conserved [19–22]. The fermionic character of two radical reactants is eliminated in reactive bonding yielding a bosonic product. As described in **Table 1**, fermionic radical-radical, SOMO-SOMO reaction yields bosonic nonradical product. Simply stated, radicals

OH) can extract 1RE from the HOMO of a singlet multiplicity

substrate). The symmetry of the reactants is preserved

*−*

is described as spin down (↓). The Pauli exclusion

<sup>−</sup> must have an *ms* = −½ (↓). Consequently, the total

is described as

<sup>−</sup> must have

H2O, but in the process

number *ms*, and only two values are allowed. When *ms* = ½, the *e*

**2.2 Bosonic versus fermionic frontier orbital interactions**

spin multiplicity with radical, paramagnetic character.

*−*

an orbital, each electron must have opposite spins, that is, one orbital *e*

spin quantum number, *S*, for a filled orbital electron-couple is ½ + −½ = 0 (↑↓).

spin up (↑); when *ms* = −½, the *e*

an *ms* = ½ (↑), the other orbital *e*

**64**

tive Fenton radical (2

nonradical substrate (1

doublet multiplicity radical (2

*Spin conservation rules.*

#### **Figure 1.**

*Triplet and electronically excited singlet molecular oxygen with emphasis on the π\* (pi antibonding) frontier orbitals. The two π\* are degenerate (same energy level). Hund's maximum multiplicity rule predicts lowest energy is achieved when each SOMO electron has the same spin, that is, the triplet state (<sup>3</sup> O2). The electronic energy of 1 O2\* is 22.5 kcal/mol (94.2 kJ/mol) above <sup>3</sup> O2.*

the bi-fermionic, bi-radical nature of <sup>3</sup> O2 that restricts its reactive potential. As per **Table 1**, the reaction of 3 O2 with a bosonic <sup>1</sup> substrate molecules is spin symmetry restricted, and could only result in the improbable generation of a bi-fermionic, triplet multiplicity product(s). However, the reaction of bi-fermionic 3 O2 with a fermionic (doublet multiplicity) radical can proceed via SOMO-SOMO overlap. As per **Table 1**, such a doublet-triplet reaction will generate a fermionic (doublet multiplicity) radical product. Thus, 3 O2 can participate in and be a necessary reactant in radical propagation reactions.

## **3. NADPH oxidase**

NADPH oxidase controls "respiratory burst" metabolism, microbicidal action, and chemiluminescence [15, 25]. The oxidase (Nox2) is a complex flavoenzyme, and a member of the Nox family of enzymes involved in various biochemical activities [26–29]. More specifically, NADPH oxidase is a flavocytochrome enzyme composed of a large membrane-bound glycoprotein (gp91phox) subunit associated with a smaller protein (p22phox). The C-terminal portion of gp91phox subunit contains the NADPH and flavin adenine dinucleotide (FAD) binding sites and an N-terminal portion that binds two heme groups. The activation of the oxidase is complex and involves other components. Association with the p67phox component is essential for full activity. The present treatment will focus on the central role of the semiquinone state of the riboflavin component of FAD and heme involvement in splitting the 2RE from <sup>1</sup> NADPH and facilitating 1RE reduction of <sup>3</sup> O2.

As illustrated in **Figure 2**, the product of 1RE reduction of <sup>3</sup> O2 is the acid hydroperoxyl radical (2 HO2) with an acid dissociation constant p*K*a of 4.8 [30]. For comparison, the p*K*a of <sup>1</sup> H2O2 is 11.7. As the pH of the phagolysosomal space approaches the p*K*a, the ratio of <sup>2</sup> HO2 to its conjugate base, the superoxide anion (2 O2 −) approaches unity, and acid disproportionation, that is, reaction of <sup>2</sup> HO2 with <sup>2</sup> O2 −, approaches maximum reaction rate. At unity, anionic repulsion is no longer a problem. The rate constant for the reaction is 4.5 × 105 M<sup>−</sup><sup>1</sup> s<sup>−</sup><sup>1</sup> at pH 7.0 and reaches a maximum of 8.5 × 107 M<sup>−</sup><sup>1</sup> s<sup>−</sup><sup>1</sup> at pH 4.8 [30, 31]. From the frontier orbital perspective, this is a SOMO-SOMO reaction that yields the nonradical (singlet multiplicity) products <sup>1</sup> H2O2 and <sup>1</sup> O2\*. As per **Table 1**, doublet-doublet annihilation yields single products [15, 32]. The reaction is sufficiently exergonic to yield <sup>1</sup> O2\* with an energy of 22.5 kcal/mol (94.1 kJ/mol) above ground state <sup>3</sup> O2.

#### **Figure 2.**

*Schema illustrating the central role of membrane-associated NADPH oxidase in respiratory burst metabolism. In the activated state, the Michaelis constant (KM) of the oxidase for NADPH is decreased. NADP+ availability controls the activities of glucose-6-PO4 dehydrogenase and 6-phosphogluconate dehydrogenase of the HMP shunt. Each pass of the cycle generates two NADPH, that is, two 2RE. In the schema, the spin multiplicities of each molecule are indicated by the superscripted number preceding the molecular description, that is, <sup>1</sup> , 2 , and 3 for singlet, doublet, and triplet multiplicity, respectively.*

**67**

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action…*

In **Figure 2**, note that all reactions in the cytoplasmic milieu are singlet multiplicity nonradical reactions and that radical production is confined to the phagolysosomal milieu. The 2RE nature of cytoplasmic redox transfer provides a bosonic

the presence of a doublet multiplicity molecule is an opportunity for SOMO-SOMO overlap. The 2RE transfer from the HOMO of a reductant to the LUMO of an

The *S* = 0 condition is described by Dirac's statement that "If a state has zero total angular momentum, the dynamical system is equally likely to have any orientation, and hence spherical symmetry occurs" [33]. In addition to providing protection from the reactive consequences of fermionic 1RE transfer in an

additional advantage. Heisenberg's uncertainty principle states that the uncertainty of momentum (Δ*p*) multiplied by the uncertainty of position (Δ*x*) is always equal to or greater than ½*ħ*, that is, Δ*p*Δ*x* ≥ ½*ħ* [17]. With regard to 2RE transfer, the bosonic orbital electron couple has *S* = 0. Consequently, the positional uncertainty of the electron-couple must be proportionally large. The *S* = 0 nature of HOMO-LUMO redox transfer involving a 2RE orbital electron-couple opens the possibility that such transfer is facilitated by quantum tunneling. The nature of such transfer would be analogous to the emission of a bosonic *S* = 0 alpha particle from an atomic

Myeloperoxidase (MPO) is a unique green cationic homo-dimeric glycosylated heme-a protein that is highly expressed in neutrophil leukocytes, making up about 5% of its dry mass [34, 35]. It is also synthesized to a lesser degree in monocytes and serves as a cellular marker for both neutrophils and monocytes. MPO synthesis occurs only during the promyelocyte phase of neutrophil development [36]. During the promyelocyte phase, MPO and other cationic lysosomal proteins are synthesized and stored in the azurophilic (aka primary) granules. Each mitotic division during the following myelocyte phase of development dilutes the azurophilic granule content per neutrophil by a half. Under normal conditions of hematopoietic production, these myelocytic phase mitoses are the rule, but under condition of neutrophil inflammatory consumption or G-CSF-stimulated marrow production, the promyelocyte pool is expanded, and there are fewer mitoses in the myelocyte phase of development. Neutrophils released into the circulation following a few days of myelopoietic stimulation show the effect of decreased myelocyte mitoses. These neutrophils are significantly increased in size due to greater azurophilic granule retention, and the MPO activity per neutrophil is severalfold higher than normal [37].

MPO, like eosinophil peroxidase, lactoperoxidase and thyroperoxidase, is a haloperoxidase (XPO). However, MPO is unique in its ability to catalyze the pHdependent oxidation of chloride [38–40]. Based on the Allen scale, fluorine (F) is the most electronegative element with a value of 4.19, followed by oxygen with a value of 3.61, then chlorine with a value of 2.87, bromine with a value of 2.69, and

With regard to chloride oxidation, the Nernstian electrochemical possibilities

O2, 2RE transfer of a bosonic orbital electron couple may have

nonradical, bosonic, and singlet multiplicity. In an atmosphere that is 20.9% 3

O2. Transfer of an orbital electron couple is

O2,

*DOI: http://dx.doi.org/10.5772/intechopen.81543*

barrier to reaction with bi-fermionic 3

atmosphere high in 3

**4. Myeloperoxidase**

oxidant maintains the bosonic *S* = 0 condition.

nucleus in alpha radiation decay [19, 20].

**4.1 Electrochemistry of halide oxidation-reduction**

iodine with a value of 2.36 [41].

and limitations are as follows [11, 42].

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action… DOI: http://dx.doi.org/10.5772/intechopen.81543*

In **Figure 2**, note that all reactions in the cytoplasmic milieu are singlet multiplicity nonradical reactions and that radical production is confined to the phagolysosomal milieu. The 2RE nature of cytoplasmic redox transfer provides a bosonic barrier to reaction with bi-fermionic 3 O2. Transfer of an orbital electron couple is nonradical, bosonic, and singlet multiplicity. In an atmosphere that is 20.9% 3 O2, the presence of a doublet multiplicity molecule is an opportunity for SOMO-SOMO overlap. The 2RE transfer from the HOMO of a reductant to the LUMO of an oxidant maintains the bosonic *S* = 0 condition.

The *S* = 0 condition is described by Dirac's statement that "If a state has zero total angular momentum, the dynamical system is equally likely to have any orientation, and hence spherical symmetry occurs" [33]. In addition to providing protection from the reactive consequences of fermionic 1RE transfer in an atmosphere high in 3 O2, 2RE transfer of a bosonic orbital electron couple may have additional advantage. Heisenberg's uncertainty principle states that the uncertainty of momentum (Δ*p*) multiplied by the uncertainty of position (Δ*x*) is always equal to or greater than ½*ħ*, that is, Δ*p*Δ*x* ≥ ½*ħ* [17]. With regard to 2RE transfer, the bosonic orbital electron couple has *S* = 0. Consequently, the positional uncertainty of the electron-couple must be proportionally large. The *S* = 0 nature of HOMO-LUMO redox transfer involving a 2RE orbital electron-couple opens the possibility that such transfer is facilitated by quantum tunneling. The nature of such transfer would be analogous to the emission of a bosonic *S* = 0 alpha particle from an atomic nucleus in alpha radiation decay [19, 20].

## **4. Myeloperoxidase**

*Neutrophils*

the bi-fermionic, bi-radical nature of <sup>3</sup>

tiplicity) radical product. Thus, 3

radical propagation reactions.

**3. NADPH oxidase**

2RE from <sup>1</sup>

products <sup>1</sup>

peroxyl radical (2

parison, the p*K*a of <sup>1</sup>

the p*K*a, the ratio of <sup>2</sup>

maximum of 8.5 × 107

H2O2 and <sup>1</sup>

O2 with a bosonic <sup>1</sup>

NADPH and facilitating 1RE reduction of <sup>3</sup>

As illustrated in **Figure 2**, the product of 1RE reduction of <sup>3</sup>

approaches unity, and acid disproportionation, that is, reaction of <sup>2</sup>

products [15, 32]. The reaction is sufficiently exergonic to yield <sup>1</sup>

lem. The rate constant for the reaction is 4.5 × 105

of 22.5 kcal/mol (94.1 kJ/mol) above ground state <sup>3</sup>

*for singlet, doublet, and triplet multiplicity, respectively.*

 M<sup>−</sup><sup>1</sup> s<sup>−</sup><sup>1</sup>

triplet multiplicity product(s). However, the reaction of bi-fermionic 3

restricted, and could only result in the improbable generation of a bi-fermionic,

fermionic (doublet multiplicity) radical can proceed via SOMO-SOMO overlap. As per **Table 1**, such a doublet-triplet reaction will generate a fermionic (doublet mul-

NADPH oxidase controls "respiratory burst" metabolism, microbicidal action, and chemiluminescence [15, 25]. The oxidase (Nox2) is a complex flavoenzyme, and a member of the Nox family of enzymes involved in various biochemical activities [26–29]. More specifically, NADPH oxidase is a flavocytochrome enzyme composed of a large membrane-bound glycoprotein (gp91phox) subunit associated with a smaller protein (p22phox). The C-terminal portion of gp91phox subunit contains the NADPH and flavin adenine dinucleotide (FAD) binding sites and an N-terminal portion that binds two heme groups. The activation of the oxidase is complex and involves other components. Association with the p67phox component is essential for full activity. The present treatment will focus on the central role of the semiquinone state of the riboflavin component of FAD and heme involvement in splitting the

**Table 1**, the reaction of 3

O2 that restricts its reactive potential. As per

O2 can participate in and be a necessary reactant in

O2.

at pH 4.8 [30, 31]. From the frontier orbital perspec-

HO2) with an acid dissociation constant p*K*a of 4.8 [30]. For com-

HO2 to its conjugate base, the superoxide anion (2

approaches maximum reaction rate. At unity, anionic repulsion is no longer a prob-

tive, this is a SOMO-SOMO reaction that yields the nonradical (singlet multiplicity)

*Schema illustrating the central role of membrane-associated NADPH oxidase in respiratory burst metabolism.* 

*controls the activities of glucose-6-PO4 dehydrogenase and 6-phosphogluconate dehydrogenase of the HMP shunt. Each pass of the cycle generates two NADPH, that is, two 2RE. In the schema, the spin multiplicities of each molecule are indicated by the superscripted number preceding the molecular description, that is, <sup>1</sup>*

*In the activated state, the Michaelis constant (KM) of the oxidase for NADPH is decreased. NADP+*

H2O2 is 11.7. As the pH of the phagolysosomal space approaches

 M<sup>−</sup><sup>1</sup> s<sup>−</sup><sup>1</sup>

O2\*. As per **Table 1**, doublet-doublet annihilation yields single

O2.

substrate molecules is spin symmetry

O2 with a

O2 is the acid hydro-

O2 −)

at pH 7.0 and reaches a

HO2 with <sup>2</sup>

O2\* with an energy

O2 −,

 *availability* 

*, 2 , and 3*

**66**

**Figure 2.**

Myeloperoxidase (MPO) is a unique green cationic homo-dimeric glycosylated heme-a protein that is highly expressed in neutrophil leukocytes, making up about 5% of its dry mass [34, 35]. It is also synthesized to a lesser degree in monocytes and serves as a cellular marker for both neutrophils and monocytes. MPO synthesis occurs only during the promyelocyte phase of neutrophil development [36]. During the promyelocyte phase, MPO and other cationic lysosomal proteins are synthesized and stored in the azurophilic (aka primary) granules. Each mitotic division during the following myelocyte phase of development dilutes the azurophilic granule content per neutrophil by a half. Under normal conditions of hematopoietic production, these myelocytic phase mitoses are the rule, but under condition of neutrophil inflammatory consumption or G-CSF-stimulated marrow production, the promyelocyte pool is expanded, and there are fewer mitoses in the myelocyte phase of development. Neutrophils released into the circulation following a few days of myelopoietic stimulation show the effect of decreased myelocyte mitoses. These neutrophils are significantly increased in size due to greater azurophilic granule retention, and the MPO activity per neutrophil is severalfold higher than normal [37].

#### **4.1 Electrochemistry of halide oxidation-reduction**

MPO, like eosinophil peroxidase, lactoperoxidase and thyroperoxidase, is a haloperoxidase (XPO). However, MPO is unique in its ability to catalyze the pHdependent oxidation of chloride [38–40]. Based on the Allen scale, fluorine (F) is the most electronegative element with a value of 4.19, followed by oxygen with a value of 3.61, then chlorine with a value of 2.87, bromine with a value of 2.69, and iodine with a value of 2.36 [41].

With regard to chloride oxidation, the Nernstian electrochemical possibilities and limitations are as follows [11, 42].

*Neutrophils*

$$\mathbf{E} = \mathbf{E\_0 - (RT/nF)} \ln{\text{ [reduced]}} / \text{[oxidized]} \tag{1}$$

where E is observed potential (in volts), E₀ is the standard potential (in volts), R is the gas constant, T is the absolute temperature, F is a faraday (23 kcal/absolute volt equivalent), and n is the number of electrons/gram equivalent transferred.

Also, appreciate that hydrogen ion concentration, [H<sup>+</sup> ], has an effect on redox chemistry.

$$\mathbf{E} = \text{(RT/F)} \,\ln\left[\text{H}^{+}\right] / \left[\text{P}\_{\text{H2}}\right]^{1/2} \tag{2}$$

PH2 is the partial pressure of H2 gas

$$\mathbf{E} = \text{(2.3RT/F)} \log \left[ \mathbf{H}^{+} \right] = \text{0.06} \log \left[ \mathbf{H}^{+} \right] = -\text{0.06} \,\text{pH} \tag{3}$$

For the reaction, Ared + Box ↔ Bred + Dox, the half reaction equations become:

$$\mathbf{E} = \mathbf{E\_0}^{\mathcal{A}} - \text{(RT/nF)} \ln \left[ \mathbf{A\_{red}} \right] / \left[ \mathbf{A\_{ox}} \right] \tag{4}$$

$$\mathbf{E} = \mathbf{E\_0}^{\tag{B}} - (\mathbf{RT}/\mathbf{n}\mathbf{F}) \ln \left[ \mathbf{B\_{red}} \right] / \left[ \mathbf{B\_{ox}} \right] \tag{5}$$

$$\mathbf{E\_{o}}^{\ \ \ \ \ \ \mathbf{B}} - \mathbf{E\_{o}}^{\ \ \ \ \ \ \text{A}} = \{ \text{RT/nF} \} \left[ \ln \left[ \mathbf{B\_{red}} \right] / \left[ \mathbf{B\_{ox}} \right] - \ln \left[ \mathbf{A\_{red}} \right] / \left[ \mathbf{A\_{ox}} \right] \right] \tag{6}$$

$$
\Delta \mathbf{E}\_{\Phi} = \text{(RT/nF)} \left[ \ln \left[ \mathbf{A}\_{\text{ox}} \right] \left[ \mathbf{B}\_{\text{red}} \right] / \ln \left[ \mathbf{A}\_{\text{red}} \right] \left[ \mathbf{B}\_{\text{ox}} \right] \right] \tag{7}
$$

$$
\Delta \mathbf{E\_{0}} = \text{(RT/nF)} \ln K\_{\text{eq}} \tag{8}
$$

*K*eq is the equilibrium constant. The change in potential (ΔE) can be expressed in terms of Gibbs free energy (ΔG).

$$
\Delta \, \mathbf{G}^{\;0} = -\text{RT} \, \ln K\_{\text{eq}} \tag{9}
$$

$$
\Delta \mathbf{G}^0 = -\mathbf{n} \mathbf{F} \,\Delta \mathbf{E}\_0 \tag{10}
$$

The schema of **Figure 3** depicts the MPO-catalyzed H2O2 oxidation of Cl<sup>−</sup> to HOCl. Chloride serves as the reductant and undergoes a 2RE oxidization yielding a chloronium intermediate (Cl+ ) that reacts with <sup>1</sup> H2O to generate hypochlorous acid with a pKa of 7.5.

$$\rm{^1Cl} \rightarrow \rm{^1Cl^\*} \star \rm{2RE} \tag{11}$$

**69**

**Figure 4.**

*(highest).*

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action…*

H2O2 + 2RE → 2<sup>1</sup>

<sup>−</sup> is relatively easy. Whereas MPO is capable of dehydrogenating Cl<sup>−</sup>, Br<sup>−</sup>, and

<sup>−</sup>, eosinophil peroxidase (EPO), lactoperoxidase, and thyroperoxidase are only

The plots of **Figure 4** illustrate that increasing acidity increases the exergonic-

import for MPO-catalyzed oxidation of chloride. Conversely, increasing alkalinity

of **Figure 4** and the chemical reaction of **Figure 5**. The Gibbs free energies shown in

*Schema depicting myeloperoxidase-catalyzed H2O2-dependent oxidation of chloride to hypochlorite, and* 

*Graph* **A** *plots changes in potential (ΔE) and graph* **B** *plots change in Gibbs free energy against pH for various halides. From bottom to top, the plotted lines represent chloride (lowest), bromide (middle) and iodide* 

H2O2-dependent oxidation of halides. This is especially

OCl<sup>−</sup>- 1

O2\* can be considered as a net disproportionation reaction, as

H2O2-driven haloperoxidase plus 1

H2O2 is the reactant common to both MPO-catalyzed reaction

*O2\*. The spin multiplicity of each molecule is indicated by the* 

The reactants and products of this MPO-catalyzed redox reaction are exclusively

As depicted in **Figure 4**, increasing acidity, that is, lowering pH, increases the ΔE (i.e., Eh2o2 − Ex<sup>−</sup>) and the Gibbs free energy for all halides. The exergonicity of MPO-catalyzed 2RE dehydrogenation of Cl<sup>−</sup> increases with increasing acidity. The required potentials for the various halides are consistent with their electronegativities. Dehydrogenation of Cl<sup>−</sup> is more difficult than Br<sup>−</sup>, but dehydrogenation of

H2O (13)

H2O2 reaction yielding 1

O2\* as

H2O2-driven

*DOI: http://dx.doi.org/10.5772/intechopen.81543*

singlet multiplicity, that is, nonradical [2, 15].

capable of dehydrogenating Br<sup>−</sup> and I<sup>−</sup>.

depicted in **Figure 5**. The combined 1

*its reaction with a second H2O2 to generate <sup>1</sup>*

*superscripted number preceding the molecular description.*

increases the exergonicity of the nonenzymatic 1

ity of MPO-catalyzed 1

OCl<sup>−</sup> generation of 1

depicted in **Figure 6**. 1

<sup>1</sup>

I

I

1

**Figure 3.**

$$\text{\textbullet } ^{1}\text{Cl}^{\*} \text{\textbullet } ^{1}\text{H}\_{2}\text{O} \rightarrow \text{\textbullet } ^{1}\text{HOCl} \text{\textbullet H}^{\*}\tag{12}$$

Note that 1 H2O2 is the oxidant for the MPO-catalyzed reaction undergoing 2RE reduction yielding two waters. One <sup>1</sup> H2O is consumed in the reaction described by Eq. (12).

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action… DOI: http://dx.doi.org/10.5772/intechopen.81543*

$$\text{\textbullet } ^1\text{H}\_2\text{O}\_2 + 2\text{RE} \rightarrow 2^1\text{H}\_2\text{O} \tag{13}$$

The reactants and products of this MPO-catalyzed redox reaction are exclusively singlet multiplicity, that is, nonradical [2, 15].

As depicted in **Figure 4**, increasing acidity, that is, lowering pH, increases the ΔE (i.e., Eh2o2 − Ex<sup>−</sup>) and the Gibbs free energy for all halides. The exergonicity of MPO-catalyzed 2RE dehydrogenation of Cl<sup>−</sup> increases with increasing acidity. The required potentials for the various halides are consistent with their electronegativities. Dehydrogenation of Cl<sup>−</sup> is more difficult than Br<sup>−</sup>, but dehydrogenation of I <sup>−</sup> is relatively easy. Whereas MPO is capable of dehydrogenating Cl<sup>−</sup>, Br<sup>−</sup>, and I <sup>−</sup>, eosinophil peroxidase (EPO), lactoperoxidase, and thyroperoxidase are only capable of dehydrogenating Br<sup>−</sup> and I<sup>−</sup>.

The plots of **Figure 4** illustrate that increasing acidity increases the exergonicity of MPO-catalyzed 1 H2O2-dependent oxidation of halides. This is especially import for MPO-catalyzed oxidation of chloride. Conversely, increasing alkalinity increases the exergonicity of the nonenzymatic 1 OCl<sup>−</sup>- 1 H2O2 reaction yielding 1 O2\* as depicted in **Figure 5**. The combined 1 H2O2-driven haloperoxidase plus 1 H2O2-driven 1 OCl<sup>−</sup> generation of 1 O2\* can be considered as a net disproportionation reaction, as depicted in **Figure 6**. 1 H2O2 is the reactant common to both MPO-catalyzed reaction of **Figure 4** and the chemical reaction of **Figure 5**. The Gibbs free energies shown in

#### **Figure 3.**

*Neutrophils*

chemistry.

E = E₀ − (RT/nF) ln [reduced]/[oxidized] (1)

where E is observed potential (in volts), E₀ is the standard potential (in volts), R is the gas constant, T is the absolute temperature, F is a faraday (23 kcal/absolute volt equivalent), and n is the number of electrons/gram equivalent transferred.

]/[PH2]

] = 0.06 log [H<sup>+</sup>

For the reaction, Ared + Box ↔ Bred + Dox, the half reaction equations become:

E = E₀ <sup>A</sup> − (RT/nF) ln [Ared]/[Aox] (4)

E = E₀ <sup>B</sup> − (RT/nF) ln [Bred]/[Box] (5)

E₀ <sup>B</sup> − E₀ <sup>A</sup> = (RT/nF) [ln [Bred]/[Box] − ln [Ared]/[Aox]] (6)

ΔE₀ = (RT/nF) [ln [Aox][Bred]/ln [Ared][Box]] (7)

ΔE₀ = (RT/nF) ln *K*eq (8)

Δ G<sup>0</sup> = −RT ln *K*eq (9)

ΔG<sup>0</sup> = −nF ΔE₀ (10)

The schema of **Figure 3** depicts the MPO-catalyzed H2O2 oxidation of Cl<sup>−</sup> to HOCl. Chloride serves as the reductant and undergoes a 2RE oxidization yielding a

) that reacts with <sup>1</sup>

H2O → <sup>1</sup>

H2O2 is the oxidant for the MPO-catalyzed reaction undergoing 2RE

Cl− → <sup>1</sup>

Cl<sup>+</sup> + <sup>1</sup>

*K*eq is the equilibrium constant. The change in potential (ΔE) can be expressed in

], has an effect on redox

1/2 (2)

] = −0.06 pH (3)

H2O to generate hypochlorous acid

Cl<sup>+</sup> + 2RE (11)

H2O is consumed in the reaction described by

HOCl + H<sup>+</sup> (12)

Also, appreciate that hydrogen ion concentration, [H<sup>+</sup>

E = (RT/F) ln [H<sup>+</sup>

PH2 is the partial pressure of H2 gas

E = (2.3RT/F) log [H<sup>+</sup>

terms of Gibbs free energy (ΔG).

chloronium intermediate (Cl+

<sup>1</sup>

reduction yielding two waters. One <sup>1</sup>

<sup>1</sup>

with a pKa of 7.5.

Note that 1

**68**

Eq. (12).

*Schema depicting myeloperoxidase-catalyzed H2O2-dependent oxidation of chloride to hypochlorite, and its reaction with a second H2O2 to generate <sup>1</sup> O2\*. The spin multiplicity of each molecule is indicated by the superscripted number preceding the molecular description.*

#### **Figure 4.**

*Graph* **A** *plots changes in potential (ΔE) and graph* **B** *plots change in Gibbs free energy against pH for various halides. From bottom to top, the plotted lines represent chloride (lowest), bromide (middle) and iodide (highest).*

**Figure 5.**

*Graph* **A** *plots changes in potential (ΔE) and graph* **B** *plots change in Gibbs free energy with respect to pH for various halides for the reaction of 1 H2O2 with 1 OCl<sup>−</sup>. In graph* **B** *the Gibbs free energies are adjusted for the 22.5 kcal mol<sup>−</sup><sup>1</sup> retained as the electronic energy of 1 O2\*, that is, the difference separating 3 O2 from 1 O3\*.*

#### **Figure 6.**

*Plot of free energy against pH for the net 1 H2O2 disproportionation reaction as described in* **Figure 2***. The free energy results are expressed with (ΔG = −27.8 kcal mol<sup>−</sup><sup>1</sup> ) and without (ΔG = −50.3 kcal mol<sup>−</sup><sup>1</sup> ) adjustment for the energy electronically conserved in oxygen excitation (ΔG = −22.5 kcal mol<sup>−</sup><sup>1</sup> ).*

**Figure 6** have been adjusted to reflect the energy conserved in electronically excited 1 O2\*. The overall net free energy is independent of the halide employed and independent of pH.

Since the reactants involved are all singlet multiplicity, the products of reaction, that is, <sup>1</sup> H2O, <sup>1</sup> Cl<sup>−</sup>, and <sup>1</sup> O2\*, are all singlet multiplicity. This provides a spin symmetry explanation as to why pouring bleach (1 OCl<sup>−</sup>) into <sup>1</sup> H2O2 causes rapid reactive release of <sup>1</sup> O2\* gas and a red chemiluminescence [23]. Caution, rapid release of gas is potentially explosive. When the concentration of <sup>1</sup> O2\* is sufficiently high, <sup>1</sup> O2\*-<sup>1</sup> O2\* collision with simultaneous relaxation yields red chemiluminescence. The relaxation of one <sup>1</sup> O2\* emits a 1270 nm photon; simultaneous relaxation of two <sup>1</sup> O2\* emits a 635 nm photon. As such, this red emission is second order with respect to <sup>1</sup> O2\*, that is, d*hν*635nm/dt = *k*[ 1 O2\*]<sup>2</sup> , and is relatively short-lived.

**71**

binding to <sup>1</sup>

damage to the <sup>1</sup>

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action…*

CO2 plus two 2RE, that is, two bosonic electron couples carried as 2NADPH. As

beads for an hour. The endoperoxide trapped indicated that at least 11.3 ± 4.9 nmol

production accounted for at least 19 ± 5% of the total oxygen consumed. Although

than expected; this study provides direct empirical evidence of significant neutro-

biological system measurements. The fact that a 1270 nm photon is measured is

**4.2 Myeloperoxidase-binding specificity focuses combustive activity**

from its point of generation. In the case of MPO generation of <sup>1</sup>

end product. They are typically microaerophilic, and often produce <sup>1</sup>

streptococci on blood agar plates results from the production of <sup>1</sup>

metabolic product. The green hemolysis associated with colonies of viridans

H2O2-producing *Strep. viridans*. Thus, LAB-produced <sup>1</sup>

microbicidal action that is restricted to the surface of the MPO-bound pathogen. MPO combustive microbicidal action is focused on the pathogen with minimum

H2O2-producing LAB, and without hemolytic damage to the added

streptococci. When a pathogen, such as *Staphylococcus aureus* or *Escherichia coli*, is contacted with a nonpathogen LAB, such as *Streptococcus viridans*, the pathogen overwhelmingly inhibits the LAB, but when a small quantity of MPO is added to a mixture, the pathogen is inhibited allowing LAB dominance. This phenomenon repeats even when erythrocytes are added to the mix at a ratio of 10 erythrocytes per bacteria. MPO selectively binds to the *S. aureus* and *E. coli* with essentially no

reactive substrates available in biological milieux, electrophilic reaction is favored

O2\* is a potent electrophilic reactant with a high probability for participation in spin-allowed reaction with electron-dense biological substrates. The lifetime

This lifetime restricts reactivity to within a radius of about 0.2–0.3 μm (microns)

MPO selectively binds to all gram-negative bacteria and most gram-positive bacteria tested, but MPO binding is weak for gram-positive lactic acid bacteria (LAB) [44, 47]. LAB are common members of the normal flora of the mouth, vagina, and colon, and include streptococci, lactobacilli, and bifidobacteria. These LAB cannot synthesize cytochromes and produce lactic acid as a metabolic

activated with phorbol-12-myristate-13-acetate (PMA), at least 14.1 ± 4.1 nmol

O2\* and two 1

glucose-6-PO4 produces 1

H2O2 for oxidation of Cl<sup>−</sup> to OCL<sup>−</sup>, and this OCL<sup>−</sup> reacts with the other

O2\* production [43]. Neutrophils were allowed to phagocytose the

neutrophils were produced. When the neutrophils were chemically

O2\* measured using this difficult trapping approach are lower

neutrophils were produced. Based on their trapping results, <sup>1</sup>

O2\*, this infrared proton emission approach is highly insensitive in

O2\* did not participate in chemical reaction. Considering the variety of

ribulose-5-PO4 plus

O2\*

O2\*, these temporal

H2O2 as a

H2O2 drives MPO

H2O2 by the

O2 in four one 1RE reduction

H2O2. As illustrated in **Figure 3**, MPO

O2\*-specific trap, for measurements

O2\* by measuring the 1270 nm near-infrared

O2 is also problematic. Although highly

O2\* restricts its reactive possibilities [44]. In

O2\* has a reactive lifetime of about 4–6 microseconds [45, 46].

O2\*. Thus, two NADPH have the potential to drive

O2\*. Steinbeck et al. have reported experiments using glass

*DOI: http://dx.doi.org/10.5772/intechopen.81543*

The double dehydrogenation of 1

steps, ultimately yielding two 1

H2O2 to generate an additional 1

the generation of three 1

illustrated in **Figure 2**, NADPH oxidase reduces four 3

beads coated with 9,10-diphenylanthracene, a <sup>1</sup>

Quantifying cellular production of <sup>1</sup>

of metastable electronically excited 1

and spatial restrictions can be advantageous.

erythrocytes, that is, no bystander injury.

O2\* relaxation to <sup>3</sup>

1

1

1

1

phil <sup>1</sup>

uses one 1

of neutrophil <sup>1</sup>

O2\*/1.25 × 106

O2\*/1.25 × 106

the quantities of <sup>1</sup>

photon emitted on <sup>1</sup>

specific for 1

proof that <sup>1</sup>

1

over relaxation.

biological milieux, <sup>1</sup>

O2\* production.

*Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action… DOI: http://dx.doi.org/10.5772/intechopen.81543*

The double dehydrogenation of 1 glucose-6-PO4 produces 1 ribulose-5-PO4 plus 1 CO2 plus two 2RE, that is, two bosonic electron couples carried as 2NADPH. As illustrated in **Figure 2**, NADPH oxidase reduces four 3 O2 in four one 1RE reduction steps, ultimately yielding two 1 O2\* and two 1 H2O2. As illustrated in **Figure 3**, MPO uses one 1 H2O2 for oxidation of Cl<sup>−</sup> to OCL<sup>−</sup>, and this OCL<sup>−</sup> reacts with the other 1 H2O2 to generate an additional 1 O2\*. Thus, two NADPH have the potential to drive the generation of three 1 O2\*. Steinbeck et al. have reported experiments using glass beads coated with 9,10-diphenylanthracene, a <sup>1</sup> O2\*-specific trap, for measurements of neutrophil <sup>1</sup> O2\* production [43]. Neutrophils were allowed to phagocytose the beads for an hour. The endoperoxide trapped indicated that at least 11.3 ± 4.9 nmol 1 O2\*/1.25 × 106 neutrophils were produced. When the neutrophils were chemically activated with phorbol-12-myristate-13-acetate (PMA), at least 14.1 ± 4.1 nmol 1 O2\*/1.25 × 106 neutrophils were produced. Based on their trapping results, <sup>1</sup> O2\* production accounted for at least 19 ± 5% of the total oxygen consumed. Although the quantities of <sup>1</sup> O2\* measured using this difficult trapping approach are lower than expected; this study provides direct empirical evidence of significant neutrophil <sup>1</sup> O2\* production.

Quantifying cellular production of <sup>1</sup> O2\* by measuring the 1270 nm near-infrared photon emitted on <sup>1</sup> O2\* relaxation to <sup>3</sup> O2 is also problematic. Although highly specific for 1 O2\*, this infrared proton emission approach is highly insensitive in biological system measurements. The fact that a 1270 nm photon is measured is proof that <sup>1</sup> O2\* did not participate in chemical reaction. Considering the variety of reactive substrates available in biological milieux, electrophilic reaction is favored over relaxation.

#### **4.2 Myeloperoxidase-binding specificity focuses combustive activity**

1 O2\* is a potent electrophilic reactant with a high probability for participation in spin-allowed reaction with electron-dense biological substrates. The lifetime of metastable electronically excited 1 O2\* restricts its reactive possibilities [44]. In biological milieux, <sup>1</sup> O2\* has a reactive lifetime of about 4–6 microseconds [45, 46]. This lifetime restricts reactivity to within a radius of about 0.2–0.3 μm (microns) from its point of generation. In the case of MPO generation of <sup>1</sup> O2\*, these temporal and spatial restrictions can be advantageous.

MPO selectively binds to all gram-negative bacteria and most gram-positive bacteria tested, but MPO binding is weak for gram-positive lactic acid bacteria (LAB) [44, 47]. LAB are common members of the normal flora of the mouth, vagina, and colon, and include streptococci, lactobacilli, and bifidobacteria. These LAB cannot synthesize cytochromes and produce lactic acid as a metabolic end product. They are typically microaerophilic, and often produce <sup>1</sup> H2O2 as a metabolic product. The green hemolysis associated with colonies of viridans streptococci on blood agar plates results from the production of <sup>1</sup> H2O2 by the streptococci. When a pathogen, such as *Staphylococcus aureus* or *Escherichia coli*, is contacted with a nonpathogen LAB, such as *Streptococcus viridans*, the pathogen overwhelmingly inhibits the LAB, but when a small quantity of MPO is added to a mixture, the pathogen is inhibited allowing LAB dominance. This phenomenon repeats even when erythrocytes are added to the mix at a ratio of 10 erythrocytes per bacteria. MPO selectively binds to the *S. aureus* and *E. coli* with essentially no binding to <sup>1</sup> H2O2-producing *Strep. viridans*. Thus, LAB-produced <sup>1</sup> H2O2 drives MPO microbicidal action that is restricted to the surface of the MPO-bound pathogen. MPO combustive microbicidal action is focused on the pathogen with minimum damage to the <sup>1</sup> H2O2-producing LAB, and without hemolytic damage to the added erythrocytes, that is, no bystander injury.

*Neutrophils*

**Figure 5.**

*22.5 kcal mol<sup>−</sup><sup>1</sup>*

*various halides for the reaction of 1*

**70**

1

dent of pH.

**Figure 6.**

tion, that is, <sup>1</sup>

short-lived.

H2O, <sup>1</sup>

*Plot of free energy against pH for the net 1*

*energy results are expressed with (ΔG = −27.8 kcal mol<sup>−</sup><sup>1</sup>*

rapid reactive release of <sup>1</sup>

neous relaxation of two <sup>1</sup>

second order with respect to <sup>1</sup>

sufficiently high, <sup>1</sup>

Cl<sup>−</sup>, and <sup>1</sup>

O2\*-<sup>1</sup>

chemiluminescence. The relaxation of one <sup>1</sup>

spin symmetry explanation as to why pouring bleach (1

*the energy electronically conserved in oxygen excitation (ΔG = −22.5 kcal mol<sup>−</sup><sup>1</sup>*

**Figure 6** have been adjusted to reflect the energy conserved in electronically excited

*Graph* **A** *plots changes in potential (ΔE) and graph* **B** *plots change in Gibbs free energy with respect to pH for* 

*OCl<sup>−</sup>. In graph* **B** *the Gibbs free energies are adjusted for the* 

*O2 from 1*

*O3\*.*

*O2\*, that is, the difference separating 3*

*H2O2 with 1*

 *retained as the electronic energy of 1*

O2\*. The overall net free energy is independent of the halide employed and indepen-

Since the reactants involved are all singlet multiplicity, the products of reac-

O2\*, that is, d*hν*635nm/dt = *k*[

rapid release of gas is potentially explosive. When the concentration of <sup>1</sup>

O2\*, are all singlet multiplicity. This provides a

*H2O2 disproportionation reaction as described in* **Figure 2***. The free* 

*) and without (ΔG = −50.3 kcal mol<sup>−</sup><sup>1</sup>*

*).*

O2\* emits a 635 nm photon. As such, this red emission is

1 O2\*]<sup>2</sup>

O2\* gas and a red chemiluminescence [23]. Caution,

O2\* collision with simultaneous relaxation yields red

OCl<sup>−</sup>) into <sup>1</sup>

O2\* emits a 1270 nm photon; simulta-

H2O2 causes

, and is relatively

O2\* is

*) adjustment for* 

#### *Neutrophils*

Specificity of MPO binding results in specificity of microbicidal action. Binding specificity allows synergistic MPO-LAB interaction and suppression of pathogens. It also suggests a role for MPO in the selection and maintenance of LAB in the normal flora [48]. Healthy human adults release about a hundred billion MPO-rich neutrophils into the circulating blood each day. The circulating lifetime of the neutrophil is reportedly less than a day. The neutrophils then leave the blood and enter a tissue and body cavity phase lasting a few days [36]. Migration of MPO-rich neutrophils into the mouth and vagina is well-known [49, 50]. When quantified, the neutrophil count of the mouth is proportional to the blood neutrophil count. These spaces typically provide an acidic milieu. Neutrophil disintegration with MPO release may provide LAB with a selective advantage in such body spaces.
