Intramolecular Electron Transfer in Yeast Flavocytochrome b2 upon One-Electron Photooxidation of the Fully Reduced Enzyme: Evidence for Redox State Control of Heme-Flavin Communication

Abstract

Flavocytochrome b2, which has been fully reduced using L-lactate, can be rapidly oxidized by 1 equiv using the laser-generated triplet state of 5-deazariboflavin. Parallel photoinduced oxidation occurs at the reduced heme and at the fully reduced FMN (FMNH2) prosthetic groups of different enzyme monomers, producing the anion semiquinone of FMN and a ferric heme. Following the initial oxidation reaction, rapid intramolecular reduction of the ferric heme occurs with concomitant oxidation of FMNH2, generating the neutral FMN semiquinone. The observed rate constant for this intramolecular electron transfer is 2200 s-1, which is 1 order of magnitude larger than the turnover number under these conditions. A slower reduction of the heme prosthetic group also occurs with an observed rate constant of approximately 10 s-1, perhaps due to intersubunit electron transfer from reduced FMN to heme. The rapid intramolecular electron transfer between the FMNH2 and ferric heme is eliminated upon addition of excess pyruvate (Ki = 3.8 mM). This latter result indicates that pyruvate inhibition of catalytic turnover apparently can occur at the FMNH2-->heme electron transfer step. These results markedly differ from those previously obtained (Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555) and confirmed here for electron transfer within the one-electron reduced enzyme and for the effect of pyruvate binding, suggesting that intramolecular communication between the heme and flavin prosthetic groups can be controlled by the redox state of the enzyme and by ligand binding to the active site.

Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase

“…An alternative hypothesis is that some coupling between the redoxstate cycling of the FAD and the open versus closed conformation stabilization was lost in the chimeric structure due to some perturbation of the interactions between the two catalytic domains. This hypothesis is supported by examples of other multidomain flavin-containing proteins, such as yeast cytochrome b 2 , which show strong conformational change on FMN reduction (Hazzard et al, 1994) or the nitric oxide synthase in which conformational changes have also been shown (Welland et al, 2008). The control of the conformational changes in multidomain electron transfer proteins by their cofactors redox states constitutes an exciting topic that can be more thoroughly investigated now that the CPR structure is available in both open and closed conformations.…”

Dynamics of flavin semiquinone protolysis in l‐α‐hydroxyacid‐oxidizing flavoenzymes – a study using nanosecond laser flash photolysis

“…Hazzard et al. [29] proposed that a neutral semiquinone is formed in flavocytochrome b 2 by electron transfer from FMNH − to heme b 2 upon back reduction of the heme after partial enzyme oxidation by the laser‐generated triplet state of 5‐deazariboflavin. However, these authors did not study the fate over time of this neutral semiquinone.…”

“…This neutral radical then undergoes deprotonation to yield the stable anionic semiquinone, previously characterized by spectrophotometry [12] and EPR [11]. Hazzard et al [29] proposed that a neutral semiquinone is formed in flavocytochrome b 2 by electron transfer from FMNH ) to heme b 2 upon back reduction of the heme after partial enzyme oxidation by the laser-generated triplet state of 5-deazariboflavin. However, these authors did not study the fate over time of this neutral semiquinone.…”

Modulation of flavocytochrome b2 intraprotein electron transfer via an interdomain hinge region

“…This describes the rate of electron transfer from fully reduced FMN haem. Reassuringly, the same rate constant, within experimental error, for this step has been directly measured by laser flash photolysis techniques, where a value of 1900p100 s −" was obtained [23]. In the case of hinge-modified flavocytochromes b # , FMN reduction is much faster than the subsequent haem reduction rate and no appreciable time lag occurs before sufficient reduced FMN is formed prior to haem reduction.…”