Resonance Raman spectra of the neutral and anionic radical semiquinones of flavin adenine dinucleotide in glucose oxidase revisited

Abstract

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 H 2 O and D 2 O 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.

Photoinduced electron transfer in glucose oxidase: a picosecond time‐resolved ultraviolet resonance Raman study

“…3 is not assignable to the UVRR band of Trp ÐC because no intense band was observed in this region of the reported spectra by Johnson et al 17 The most probable assignment is that the band is due to FAD Ð . Schelvis et al 18 reported visible RR spectra of FAD in GOD, which exhibited a prominent RR band at 1330 cm 1 . Figure 4 shows the picosecond time-resolved UVRR spectra of GOD measured with the 240-nm probe pulse.…”

Recent Advances in linear and nonlinear Raman spectroscopy I

“…Asakawa et al 281 have applied a previously developed Raman linear intensity difference method to study Trp189 in bacteriorhodopsin, a transmembrane protein which acts as a light-driven proton pump. Finally, Schelvis et al 283 revisited the resonance Raman spectra of the neutral and anionic radical semiquinones of flavin adenine dinucleotide in glucose oxidase. In order to characterize the protein structure of human myeloperoxidase and its Met243Thr and Asp94Val mutants Brogioni et al 282 have undertaken a comprehensive resonance Raman study as a function of excitation wavelength and incident laser polarization.…”