**1. Introduction**

300 Thermodynamics – Interaction Studies – Solids, Liquids and Gases

[4] Atkinson C, Petreanu S, Clark NN, Atkinson RJ etc. Numerical simulation of a twostroke engine-alternator combination. SAE Technical Paper 1999-01-0921, 1999 [5] Shoukry E, Taylor S, Clark N. Numerical simulation for parametric study of a two-stroke

[6] Max E. FPEC, Free piston energy converter. In Proceedings of the 21st Electric Vehicle

[7] Blarigan PV, Paradiso N, Goldsborough SS. Homogeneous charge compression ignition with

[8] Blarigan PV. Advanced internal combustion electrical generator. Proceedings of the 2002

[9] Fredrisksson J, Denbratt I. Simulation of a two-stroke free piston engine. SAE paper 2004-

[10] Nemecek P, Vysoky O. Control of two-stroke free-piston generator. Proceeding of the

[11] Mikalsen R, Roskilly AP. The design and simulation of a two-stroke free piston engine

[12] Mikalsen R, Roskilly AP. A computational study of free-piston diesel engine combustion, Appl Energ (2008), doi: 10.1016/j.apenergy.2008.08.004 [13] Xiao J et al. Motion characteristic of a free piston linear engine. Appl Energy (2009),

[14] Cawthorne WR, Famouri P, Chen JD. Development of a linear alternator-engine for hybrid electric vehicle application. IEEE transactions on vehicular technology, vol.48, NO.6, 1999

[15] Wang JB, Howe H. A linear permanent magnet generator for a free-piston energy

[16] Deng Z, Bold I, Nasar SA. Fields in permanent magnet linear synchronous machines.

[17] Němeček P, Vysoký O. Modeling and control of free-piston generator. IFAC

[18] Caresana F, Comodi G, Pelagalli L. Design approach for a two-stroke free piston engine for

[19] Hohenberg GF. Advanced approaches for heat transfer calculations. SAE Special

[20] Stone R. Introduction to internal combustion engine. ISBN 0-7680-0495-0, Society of

[21] Nagy CT. Linear engine development for series hybrid electrical vehicles. Dissertation,

[22] Buckingham, Edgar (1914). On Physically Similar Systems: Illustrations of the Use of Dimensional Analysis. Phys. Rev. 4: 345. doi:10.1103/PhysRev.4.345 [23] Goldsborough SS, Blarigan PV. A numerical study of a free piston IC engine operating

[24] Goldsborough SS, Blarigan PV. Optimizing the scavenging system for a two-stroke

[25] Bergman M, Fredriksson J, Golovitchev VI. CFD-Base Optimization of a Diesel-fueled

IEEE Transactions on magnets. Vol. MAG-22, NO.2, 1986

Mechatronic systems, Sydney, Australia, 2004

Automotive Engineers, Inc. Warrendale, Pa, 1999

West Virginia: West Virginia University, 2004

Publications. SP-449, pp. 61-79, 1979

at the SAE Congress 2003, 2003

01-2423, 2008

a free piston: A new approach to ideal Otto cycle performance. SAE paper 982484, 1998

for electric power generation. Appl. Therm. Eng. (2007), doi:

converter. 2005 IEEE International Conference on Electric Machines and Drives,

electric power generation. Society of Automotive Engineers of Japan 2004-32-0037, 2004

on homogeneous charge compression ignition combustion. SAE paper 990619, 1999

cycle, free piston engine for high efficiency and low emissions: A computational approach. International Multidimensional Engine Modeling User's Group Meeting

Free Piston Engine Prototype for Conventional and HCCI Combustion. SAE 2008-

direct injection linear engine. SAE paper 2002-01-1739, 2002

U.S. hydrogen program review, NREL/CP-610-32405, 2002

Symposium & Exhibition, EVS21, Monaco, 2005

01-1871, 2004

p1521-1528, 2005

6th Asian control conference, 2006

10.1061/j.applthermaleng. 2007.04.009

doi:10.1016/j.apenergy.2009.07.005

Ligand-induced conformational transitions play an eminent role in the biological activity of proteins including recognition, signal transduction, and membrane trafficking. Conformational transitions occur over a broad time range starting from picosecond transitions that reflect reorientation of amino acid side chains to slower dynamics on the millisecond time-scale that correspond to larger domain reorganization (Henzler-Wildman et al., 2007). Direct characterization of the dynamics and energetics associated with conformational changes over such a broad time range remains challenging due to limitations in experimental protocols and often due to the absence of a suitable molecular probe through which to detect structural reorganization. Photothermal methods such as photoacoustic calorimetry (PAC) and photothermal beam deflection provide a unique approach to characterize conformational transitions in terms of time resolved volume and enthalpy changes (Gensch&Viappiani, 2003; Miksovska&Larsen, 2003). Unlike traditional spectroscopic techniques that are sensitive to structural changes confined to the vicinity of a chromophore, photothermal methods monitor overall changes in volume and enthalpy allowing for the detection of structural transitions that are spectroscopically silent (i.e. do not lead to optical perturbations of either intrinsic or extrinsic chromophores).

Myoglobin (Mb) and hemoglobin (Hb) play a crucial role in the storage and transport of oxygen molecules in vertebrates and have served as model systems for understanding the mechanism through which protein dynamics regulate ligand access to the active site, ligand affinity and specificity, and, in the case of hemoglobin, oxygen binding cooperativity. Mb and individual α- and β- subunits of Hb exhibit significant structural similarities, i.e. the presence of a five coordinate heme iron with a His residue coordinated to the central iron (proximal ligand) and a characteristic "3-on-3" globin fold (Fig. 1)(Park et al., 2006; Yang&Phillips Jr, 1996). Both proteins reversibly bind small gaseous ligands such as O2, CO, and NO. The photo-cleavable Fe-ligand bond allows for the monitoring of transient deoxy intermediates using time-resolved absorption spectroscopy (Carver et al., 1990; Esquerra et al., 2010; Gibson et al., 1986) and time resolved X-ray crystallography (Milani et al., 2008; Šrajer et al., 2001). Based on spectroscopic data and molecular dynamics approaches (Bossa et al., 2004; Mouawad et al., 2005), a comprehensive molecular mechanism for ligand migration in Mb was proposed including an initial diffusion of the photo-dissociated CO molecule into the internal network of hydrophobic cavities, followed by a return

Time Resolved Thermodynamics Associated with Diatomic Ligand Dissociation from Globins 303

Mb, Hb, inositol hexakisphosphate (IHP), and bezafibrate (BZF) were purchased from Sigma-Aldrich and used as received. Fe(III) tetrakis(4-sulfonatophenyl)porphine (Fe(III)4SP) was obtained from Frontier-Scientific Inc. Oxymyoglobin (O2-Mb) samples were prepared by dissolving the protein in 50 mM HEPES buffer pH 7.0. The sample was then purged with Ar for 10 min and reduced by addition of a freshly prepared solution of sodium dithionite. The quality of the deoxymyoglobin (deoxyMb) was verified by UV-visible spectroscopy. (O2-Mb) was obtained by bubbling air through deoxyMb sample. The CO bound hemoglobin sample was prepared by desolving Hb in 100 mM HEPES buffer pH 7.0 in a 0.5 x 1cm quartz cuvette. The concentration of allosteric effectors was 5 mM for BZF and 1 mM for IHP. The sample was then sealed with a septum cap and purged with Ar for 10 min, reduced with a small amount of sodium dithionite to prepare deoxyhemoglobin (deoxyHb), and subsequently bubbled with CO for approximately 1 min. Preparation of O2-Mb and CO-

The quantum yield () was determined as described previously (Belogortseva et al., 2007). All transient absorption measurements were carried out on 50 µM samples in 50 or 100 mM HEPES buffer, pH 7.0, placed in a 2 mm path quartz cell. The cell was placed into a temperature controlled holder (Quantum Northwest) and the ligand photo-dissociation was triggered using a 532 nm output from a Nd:YAG laser (Minilite II, Continuum). The probe beam, an output from the Xe arc lamp (200 W, Newport) was propagated through the center of the cell and then focused on the input of a monochromator (Yvon-Jovin ). The intensity of the probe beam was detected using an amplified photodiode (PDA 10A, Thornlabs) and subsequently digitized (Wave Surfer 42Xs, 400 MHz). The power of the pump beam was kept below 50 µJ to match the laser power used in photoacoustic measurements. The quantum yield was determined by comparing the change in the sample absorbance at 440 nm with that of the reference, CO bound myoglobin of known quantum

> Φ = �������������� ����������

where ΔAsam and ΔAref are the absorbance change of the sample and reference at 440 nm, respectively, and Δsam and Δref are the change in the extinction coefficient between the CO

(1)

Hb aducts was checked by UV-vis spectroscopy (Cary50, Varian).

yield (ref= 0.96 (Henry et al., 1983)) according to Eq 1:

bound and reduced form of the sample and the reference, respectively.

Scheme 1.

**2. Material and methods** 

**2.1 Quantum yield determination** 

Fig. 1. Left: Ribbon representation of the tetrameric human Hb structure (PBD entry 1FDH). Right: horse heart Mb structure (PDB entry 1WLA). The heme prosthetic groups are shown as sticks. In the case of Mb, the distal and proximal histidine are visualized.

into the distal pocket and subsequent rebinding to heme iron or escape from the protein through a distal histidine gate. The ligand migration into internal cavities induces a structural deformation, which promotes a transient opening of a gate in the CO migration channel. Such transitional reorganization of the internal cavities is ultimately associated with a change in volume and/or enthalpy and thus can be probed using photothermal techniques. Indeed, CO photo-dissociation from Mb has been intensively investigated using PAC by our group and others (Belogortseva et al., 2007; Peters et al., 1992; Vetromile, et al., 2011; Westrick&Peters, 1990; Westrick et al., 1990) and these results lead to a thermodynamic description of the transient "deoxy intermediate" that is populated upon CO photo-dissociation.

The mechanism of ligand migration in Hb is more complex, since it is determined by the tertiary structure of individual subunits as well as by the tetramer quaternary structure. Crystallographic data have shown that the structure of the fully unliganded tense (T) state of Hb and the fully ligated relaxed (R) states differ at both the tertiary and quaternary level (Park et al., 2006). Crystallographic and NMR studies suggest that the fully ligated relaxed state corresponds to the ensemble of conformations with distinct structures (Mueser et al., 2000; Silva et al., 1992). Moreover, Hb interactions with diatomic ligands is modulated by physiological effectors such as protons, chloride, and phosphate ions, and non-physiological ligands including inositol hexakisphosphate (IHP) and bezafibrate (BZF) (Yonetani et al., 2002). Despite a structural homology between Hb and Mb, the network of internal hydrophobic cavities identified in Mb is not conserved in Hb suggesting distinct ligand migration pathways in this protein (Mouawad et al., 2005; Savino et al., 2009). Here we present thermodynamic profiles of CO photo-dissociation from human Hb in the presence of heterotropic allosteric effectors IHP and BZF. In addition, we include an acoustic study of oxygen photo-dissociation from Mb that has not been investigated previously using photothermal methods, despite the fact that oxygen is the physiological ligand for Mb.

Scheme 1.

302 Thermodynamics – Interaction Studies – Solids, Liquids and Gases

Fig. 1. Left: Ribbon representation of the tetrameric human Hb structure (PBD entry 1FDH). Right: horse heart Mb structure (PDB entry 1WLA). The heme prosthetic groups are shown

into the distal pocket and subsequent rebinding to heme iron or escape from the protein through a distal histidine gate. The ligand migration into internal cavities induces a structural deformation, which promotes a transient opening of a gate in the CO migration channel. Such transitional reorganization of the internal cavities is ultimately associated with a change in volume and/or enthalpy and thus can be probed using photothermal techniques. Indeed, CO photo-dissociation from Mb has been intensively investigated using PAC by our group and others (Belogortseva et al., 2007; Peters et al., 1992; Vetromile, et al., 2011; Westrick&Peters, 1990; Westrick et al., 1990) and these results lead to a thermodynamic description of the transient "deoxy intermediate" that is populated upon

The mechanism of ligand migration in Hb is more complex, since it is determined by the tertiary structure of individual subunits as well as by the tetramer quaternary structure. Crystallographic data have shown that the structure of the fully unliganded tense (T) state of Hb and the fully ligated relaxed (R) states differ at both the tertiary and quaternary level (Park et al., 2006). Crystallographic and NMR studies suggest that the fully ligated relaxed state corresponds to the ensemble of conformations with distinct structures (Mueser et al., 2000; Silva et al., 1992). Moreover, Hb interactions with diatomic ligands is modulated by physiological effectors such as protons, chloride, and phosphate ions, and non-physiological ligands including inositol hexakisphosphate (IHP) and bezafibrate (BZF) (Yonetani et al., 2002). Despite a structural homology between Hb and Mb, the network of internal hydrophobic cavities identified in Mb is not conserved in Hb suggesting distinct ligand migration pathways in this protein (Mouawad et al., 2005; Savino et al., 2009). Here we present thermodynamic profiles of CO photo-dissociation from human Hb in the presence of heterotropic allosteric effectors IHP and BZF. In addition, we include an acoustic study of oxygen photo-dissociation from Mb that has not been investigated previously using photothermal methods, despite the fact that oxygen is

as sticks. In the case of Mb, the distal and proximal histidine are visualized.

CO photo-dissociation.

the physiological ligand for Mb.
