Proton-Coupled Electron Transfer of Flavodoxin Immobilized on Nanostructured Tin Dioxide Electrodes:  Thermodynamics versus Kinetics Control of Protein Redox Function

Abstract

In this paper, we report a spectroelectrochemical investigation of proton-coupled electron transfer in flavodoxin D. vulgaris Hildenborough (Fld). Poly-L-lysine is used to promote the binding of Fld to the nanocrystalline, mesoporous SnO(2) electrodes. Two reversible redox couples of the immobilized Fld are observed electrochemically and are assigned by spectroelectrochemistry to the quinone/semiquinone and semiquinone/hydroquinone couples of the protein's flavin mononucleotide (FMN) redox cofactor. Comparison with control data for free FMN indicates no contamination of the Fld data by dissociated FMN. The quinone/semiquinone and semiquinone/hydroquinone midpoint potentials (E(q/sq) and E(sq/hq)) at pH 7 were determined to be -340 and -585 mV vs Ag/AgCl, in good agreement with the literature. E(q/sq) exhibited a pH dependence of 51 mV/pH. The kinetics of these redox couples were studied using cyclic voltammetry, cyclic voltabsorptometry, and chronoabsorptometry. The semiquinone/quinone reoxidation is found to exhibit slow, potential-independent but pH-sensitive kinetics with a reoxidation rate constant varying from 1.56 s(-)(1) at pH 10 to 0.0074 s(-)(1) at pH 5. The slow kinetics are discussed in terms of a simple kinetics model and are assigned to the reoxidation process being rate limited by semiquinone deprotonation. It is proposed that this slow deprotonation step has the physiological benefit of preventing the undesirable loss of reducing equivalents which results from semiquinone oxidation to quinone.

Impact of the N5-proximal Asn on the Thermodynamic and Kinetic Stability of the Semiquinone Radical in Photolyase

“…In flavodoxins, this residue is a Gly that orients its backbone carbonyl to provide an H-bond acceptor to the protonated flavin N5 of the hq and neutral sq radical (7)(8)(9). Complete oxidation requires deprotonation, and the Gly flips to offer its backbone NH as an Hbond donor to N5.…”

Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones

“…Assuming active microbial metabolism, the value for the back reaction should be negligible, because the concentration of oxidized heme would be markedly lowered by the continuous supply of respiratory electrons to the OmcA-MtrCAB complex (32). We calculated the k f value as a function of the electron-donor potential for both reactions 1 and 2 using plausible α values (α = 0.4, 0.5, and 0.6) and the reported k 0 values for the electrode reaction of flavins, where k 0 (Ox/Sq) = 0.1 and k 0 (Ox/Hq) = 0.15 (33,34) (Fig. S7A).…”

Structural Basis of Biological NO Generation by Octaheme Oxidoreductases

“…The electrode was composed of a mesoporous, nanocrystalline layer of SnO 2 coated onto a glass slide (32). The electrode (ϳ0.7-cm 2 geometric area) was covered with 5 l of 3.5 mg ml Ϫ1 kustc1061 in potassium P i buffer and incubated on ice for 10 min.…”