Johannes P. M. Schelvis

Flavin radical semiquinones are intermediates in important physiological processes. Resonance Raman (RR) spectroscopy is an important tool to determine the interactions between these radical intermediates and their protein environment that regulate their reactivity and role in the reaction mechanisms. RR spectra of flavin radical semiquinones have been available for several flavoproteins, and those in the glucose oxidase (GO) seem significantly different from all the other available data. Since GO is often used not only as a standard for flavin‐containing proteins but also in biotechnological applications, we decided to reexamine the RR spectra of the neutral and anionic radical semiquinone forms of the flavin adenine dinucleotide (FAD) cofactor in this enzyme. The new data show that the vibrational wavenumbers of the neutral and anionic radical semiquinone forms of FAD in GO are very similar to those in other flavoproteins. The discrepancies that were observed earlier seem related to contributions of the FAD in different redox and protonation states. We also obtained the first RR spectra of the oxidized FAD cofactor in GO. Analysis of the vibrations of the oxidized FAD and its anionic radical semiquinone in GO in H₂O and D₂O solutions indicates that the subtle differences between these spectra in GO and in other flavoproteins are related to the weak hydrogen‐bonding environment of the FAD cofactor in GO. Copyright © 2006 John Wiley & Sons, Ltd.

We report the first low-frequency resonance Raman spectra of ferric endothelial nitric oxide synthase (eNOS) holoenzyme, including the frequency of the Fe-S vibration in the presence of the substrate L-arginine. This is the first direct measurement of the strength of the Fe-S bond in NOS. The Fe-S vibration is observed at 338 cm(-1) with excitation at 363.8 nm. The assignment of this band to the Fe-S stretching vibration was confirmed by the observation of isotopic shifts in eNOS reconstituted with 54Fe- and 57Fe-labeled hemin. Furthermore, the frequency of this vibration is close to those observed in cytochrome P450(cam) and chloroperoxidase (CPO). The frequency of this vibration is lower in eNOS than in P450(cam) and CPO, which can be explained by differences in hydrogen bonding to the proximal cysteine heme ligand. On addition of substrate to eNOS, we also observe several low-frequency vibrations, which are associated with the heme pyrrole groups. The enhancement of these vibrations suggests that substrate binding results in protein-mediated changes of the heme geometry, which may provide the protein with an additional tuning element for the redox potential of the heme iron. The implications of our findings for the function of eNOS will be discussed by comparison with P450(cam) and model compounds.