Protein-ligand interactions in lumazine protein and in Desulfovibrio flavodoxins from resonance coherent anti-Stokes Raman spectra

Abstract

The resonance coherent anti-Stokes Raman technique was used to obtain vibrational spectra of flavin in flavodoxins from Desulfovibrio gigas and Desulfovibrio vulgaris and of the simpler 6,7-dimethyl-8-ribityllumazine chromophore in the blue fluorescence lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. In the region examined, 1100-1700 cm-1, the Raman spectrum of the lumazine is less crowded than that of the flavin and this facilitates assignment of observed frequencies to particular vibrational modes. Certain modes are not affected by chromophore binding to the protein, but others are changed in frequency or intensity in a way that can be rationalized by expected interactions of the chromophore with the amino acid residues of the binding site. For example, a tentative assignment of change in hydrogen bonding at N(5) is suggested as the cause of the frequency shift for the chromophore in both flavodoxins (free-bound, 1582-1572 cm-1) and for C(4)=O in glucose oxidase (1359-1364 cm-1) and lumazine protein (1359-1362 cm-1). Shifts of the C(2)-N(3) mode in D2O may arise from hydrogen-bonding changes at C(2)=O in lumazine protein (1284-1291 cm-1), flavodoxin (1300-1280 cm-1), and glucose oxidase (1297-1287 cm-1). Bonding at N(3)-H may be the origin of changes in the frequency or intensity of the amide III mode in riboflavin binding protein and glucose oxidase. A stacking interaction is suggested for the change in a pyrimidine ring mode in FAD (1508 cm-1) since this mode is found at 1504 cm-1 in 30% Me2SO/H2O, where the adenine and pyrimidine are unstacked. The results clearly indicate different interactions in the binding sites of those proteins studied to date.

Coherent anti‐stokes raman spectra of isoalloxazines

“…This idea of 'localization' is brought out in the recent semi-empirical analysis of the isoalloxazine vibrations by Bowman and Spiro. 6 The points around the ring that have the potential for hydrogen bonding are the nitrogens at N(1), N(3) and N(5) and the carbonyls (2) and (4). If this potency of hydrogen bonding is mimicked by isotopic substitution at these positions, then we can conclude that such bridging would lead to measureable downshifts in certain frequencies and that certain bands would be more susceptible to changes depending on the position of isotopic substitution.…”

“…The band is sensitive to heavy atom substitution at C(4a) and C (4,lOa). Especially the intensity is affected by isotopic substitution at C (4,lOa).…”

“…Especially the intensity is affected by isotopic substitution at C (4,lOa). This points to a fair amount of framework contribution in the CH3-bending mode.…”

Time-resolved fluorescence studies of flavodoxin. Fluorescence decay and fluorescence anisotropy decay of tryptophan in Desulfovibrio flavodoxins

“…The chemical and physical properties of flavodoxins, isolated from various organisms, have been extensively investigated with several spectroscopic techniques (Edmondson and Tollin 1971;D'Anna and Tollin 1972;Ryan and Tollin 1973;Eaton etal. 1975;Visser et al 1977Visser et al , 1980Visser et al a, 1987Irwin et al 1980;Vervoort et al 1985Vervoort et al , 1986. The crystal structures of a few flavodoxins in different redox states have been reported at high resolution (Watenpaugh et al 1973;Burnett et al 1974;Smith et al 1983).…”

Time‐resolved fluorescence studies of flavodoxin Demonstration of picosecond fluorescence lifetimes of FMN in Desulfovibrio flavodoxins

“…Optical studies have been summarized in [2]. Raman spectra turned out to be characteristic for the flavodoxin species studied Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznari, Poland [5,6]. Recent NMR data provided strong support for tightly associated FMN in a compact flavodoxin conformation [7].…”