**3. Early applications to studying subtle aspects of protein/enzyme structure**

The spin label method is a reporter group technique, a concept in the 1960s [7], as depicted in Fig. 4.

**Figure 4.** Schematic representation of an enzyme-substrate complex in native protein (top left), protein containing reporter group (solid black area) adjacent to substrate binding area (top right), and reporter group distant from substrate binding area (bottom right). From [7] with permission

The revolutionary developments in organic synthesis of nitroxide spin labels helped us overcome a major challenge for biochemical studies, where the plan was to attempt to fit the spin label to the biological system in as subtle a manner as possible, that is to "fool" the system into thinking it was binding to, or interacting with, a real, natural substrate or cofactor. One of the early attempts in the McConnell laboratory was the synthesis, a nitrophenylester of 1-carboxyl-2,2,5,5-tetramethylpyrollidine, depicted in Figure 5 (left), so that one could take advantage of the esterase activity of the enzyme α–chymotrypsin and virtually 'hook' the enzyme during its action on the compound. The approach was to isolate the active acyl-enzyme intermediate, covalently attached at serine 195, which is where the intermediate in the enzyme catalyzed hydrolysis resides. Indeed, the spin labeled acylenzyme intermediate reflected a tightly bound (possibly rigid, uniquely oriented) spin label at the active site [8]. However, it became much more difficult when one wanted details as to how the enzyme handled this spin labeled substrate analog and business area to do single crystal studies of the spin labeled chymotrypsin order to derive information about the precise orientation of the label. This could be gleaned from knowledge of the anisotropic hyperfine constants and anisotropic g-factors. One could determine orientation of the N-O bond and work backwards to find the orientation of the nitroxide five-membered ring on the protein. With knowledge of the x-ray structure of α-chymotrypsin and its reactive intermediate structures already known, the process was straightforward, Bauer and Berliner were able to obtain individual binding orientation of the R-and S-enantiomers of this particular house substrate panel and from that understand why the more slowly released enantiomer substrate acyl group was mis-oriented at the active site making hydrolysis by an activated water molecule quite difficult [9].

6 Nitroxides – Theory, Experiment and Applications

IUPAC rules.

**structure** 

in Fig. 4.

Hence, the most inappropriate term for these radicals, nitroxides, has been used most widely and, as of 2009, has been cited about 115,000 times, nitroxyl about 29,500 times, iminoxyl (initiated by E.G. Rozantsev and coworkers) about 4,150 times. Aminoxyl, the most correct, has been cited 3,910 times. Obviously, the term nitroxyl is way out of line, pushed only bnefly by the late Andre Rassat, but is not relevant to this class of radicals (although I have two colleagues who continue to propogate this misuse!) I recall a friendly conversation with my long time colleague, Jim Hyde, who emphasized that if it becomes common usage, it's here so stay and to just give up on the issue. However when we academics teach organic chemistry to our young students, we try to imbue them with the correct terminology. Furthermore, standard states and nomenclature were designed so that scientists in the world can understand one another. It is clear that the correct nomenclature that the spinlabel community should be using is aminoxyl radicals. It would be great if, from this point in our history moving forward, we might correct this error in the future and abide by the

**3. Early applications to studying subtle aspects of protein/enzyme** 

The spin label method is a reporter group technique, a concept in the 1960s [7], as depicted

**Figure 4.** Schematic representation of an enzyme-substrate complex in native protein (top left), protein containing reporter group (solid black area) adjacent to substrate binding area (top right), and reporter

The revolutionary developments in organic synthesis of nitroxide spin labels helped us overcome a major challenge for biochemical studies, where the plan was to attempt to fit the spin label to the biological system in as subtle a manner as possible, that is to "fool" the system into thinking it was binding to, or interacting with, a real, natural substrate or cofactor. One of the early attempts in the McConnell laboratory was the synthesis, a nitrophenylester of 1-carboxyl-2,2,5,5-tetramethylpyrollidine, depicted in Figure 5 (left), so that one could take advantage of the esterase activity of the enzyme α–chymotrypsin and

group distant from substrate binding area (bottom right). From [7] with permission

**Figure 5.** (left). Spin-labeled substrate (R,S)-2,2,5,5-tetramethyl-3-carboxy-pyrrolidine-p-nitrophenyl ester (a). The acyl-nitroxide group that is covalently linked to Ser 195 of α-chymotrypsin (b). It was later found that the "specificity" for a spin-labeled acyl-chymotrypsin was the S- enantiomer, although both enantiometers can be isolated as acyl enzymes. (right) Chemical structure of SL-NAD+ ,nicotinamide N6- ([15N,2H17]2,2,6,6-tetramethylpiperidine-4-yl-l-oxyl)adenine nucleotide [10]

The real sophistication came in studies of enzymes that bound nucleotide analogues or, in fact, DNA and nucleotide complexes. In some beautiful work Trommer and colleagues synthesized NAD analog, SL-NAD+, where the nitroxide ring was fused onto the nicotinamide, the structure, shown above in Figure 5b (right) [10]. The enzyme bound very tightly to this NAD analog and its precise orientation could be determined. What was interesting was that in the example glyceraldehyde phosphate dehydrogenase, a tetrameric enzyme that binds one NAD per subunit in each tetramer, the distance between two NAD spin labeled analogs could be determined from the electron-electron dipole interaction. This was the first example of distance measurements involving two spin labels within a protein structure and, due to the fortuitous situation of a perfectly, rigidly bound spin label, distances could be determined precisely [10]. This study still remains the gold standard of distance measurements by electron-electron dipolar interactions.
