[en] Flash photolysis techniques have been used to study the effect of 8α-substitution on flavin triplet state formation and decay and on the properties of neutral and anionic semiquinones. Compared with riboflavin, the N(1) and N(3) isomers of 8α-histidylriboflavin showed a lower triplet yield (approximately 10%) and a faster rate of decay (approximately 4-fold). Acetylation of the histidyl α-amino groups and of the ribityl side chain resulted in a 2-fold increase in triplet yield and a 2-fold slower rate of decay. The yield of neutral 8α-substituted flavin semiquinones upon flash photolysis in the presence of EDTA was approximately 50% that given by riboflavin. These substituted flavin neutral semiquinones dismutated at a rate 2 to 3 times slower than the corresponding unsubstituted form, although the anionic semiquinones dismutated at approximately the same rate. In the presence of oxygen, the kinetics of semiquinone decay changed from second order to pseudo-first order upon raising the pH, thus showing anionic semiquinone oxidation as seen previously with unsubstituted flavins. The pK values for the ionization of the neutral 8α-substituted flavin semiquinones were 1 to 1.5 units lower than the unsubstituted form. The anionic 8α-substituted flavin semiquinones reacted with oxygen at a rate 2 to 10 times more slowly than did the riboflavin form. Such alterations in properties probably reflect the electron-withdrawing effect of the 8α-substituents on the flavin ring system. (author)

[en] A method is described for synthesis of riboflavin selectively labeled in the hydrogens at the 5'-hydroxymethyl position. In this method, a vitamin B₂-aldehyde-forming enzyme from Schizophyllum commune is used to specifically and completely oxidize the 5'-hydroxymethyl of riboflavin to the 5'-aldehyde. This reaction is monitored spectrophotometrically by the reduction of 2,6-dichlorophenolindophenol at 600 nm. Appearance of aldehyde product was directly quantitated by reverse-phase high-performance liquid chromatography. Product is extracted from the incubation mixture by phenol after saturation with (NH₄)₂SO₄ and then further purified by benzyl alcohol extraction. The 5'-aldehyde is reduced with appropriately labeled sodium borohydride to yield the vitamin specifically labeled in the 5'-hydroxymethyl group. (author)

[en] The desulfo-inhibited Mo(V) center of bovine milk xanthine oxidase has been investigated by electron-nuclear double resonance spectroscopy. Comparison of spectral data obtained from samples prepared with [1H4]ethylene glycol and with [2H4]ethylene glycol allowed assignment of proton resonance lines due to the methylene protons of the coordinated ethylene glycol (AH = 3.6 MHz). Deuterium resonance lines were observed with the deuterated sample (AD = 0.4 MHz). No spectral evidence was obtained for any weakly coupled nitrogen nuclei to the Mo center under a variety of conditions. Dissolution of the sample in D2O had little effect on the resonance lines centered about the proton Zeeman frequency, which shows they are not due to exchangeable protons and suggests the Mo center does not have contact with bulk solvent. A deuterium delta m = +/- 2 forbidden transition is observed at high radio-frequency power levels, which suggests either an exchangeable proton on a Mo ligand or a coordinated solvent. Weakly coupled, nonexchangeable proton lines are observed about the free proton frequency, which exhibit properties characteristic of alpha-protons. A number of arguments are presented to support the proposal that these protons originate from the C(1') and C(2') positions on the side chain of the molybdopterin cofactor

[en] The effect of isotopic substitution of the 8-H of xanthine (with ²H and ³H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in ^H/T(V/K) values was observed between xanthine oxidase and xanthine dehydrogenase. Xanthine dehydrogenase exhibited a larger ^T/D(V/K) value than that observed for xanthine oxidase. Observed ^H/T(V/K) values for either enzyme are less than those ^H/T(V/K) values calculated with ^D/T(V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8-³H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (C_r) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect (^Dk) for xanthine oxidation is calculated. By the use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be ∼75-fold faster than k_cat for xanthine oxidase and ∼10-fold faster than k_cat for xanthine dehydrogenase. Values calculated for each enzyme were found to be identical within experimental uncertainty

[en] Circular dichroism and ¹H and ³¹P nuclear magnetic resonance spectroscopy have been used to investigate complex formation between cytochrome c and the flavodoxins from Azotobacter vinelandii and Clostridium pasteurianum. Such complexes are known to be involved in the mechanism of electron transfer between these two redox proteins. A large increase in ellipticity in the Soret band of the cytochrome heme was observed upon formation of the Clostridium flavodoxin complex, whereas much smaller changes were found for the complexes with either Azotobacter flavodoxin or an 8α-imidazolyl-FMN-substituted Clostridium flavodoxin analogue. Similarly, the magnitudes of the perturbations of the contact-shifted heme proton resonances obtained upon complexation of cytochrome c by Azotobacter flavodoxin were much smaller than those previously shown for Clostridium flavodoxin. ³¹P nuclear magnetic resonance measurements were also consistent with differences in the interactions between the components in the complexes of the two flavodoxins with cytochrome c. It is suggested that these spectral changes are due to a loosening or opening of the heme crevice upon Clostridium flavodoxin binding, which allows closer contact between the heme and flavin prosthetic groups and results in a faster rate of electron transfer. The implications of these observations for biological oxidation-reduction processes are considered