Direct electrochemical analyses of human cytochromes b5 with a mutated heme pocket showed a good correlation between their midpoint and half wave potentials

Abstract

BackgroundCytochrome b5 performs central roles in various biological electron transfer reactions, where difference in the redox potential of two reactant proteins provides the driving force. Redox potentials of cytochromes b5 span a very wide range of ~400 mV, in which surface charge and hydrophobicity around the heme moiety are proposed to have crucial roles based on previous site-directed mutagenesis analyses.MethodsEffects of mutations at conserved hydrophobic amino acid residues consisting of the heme pocket of cytochrome b5 were analyzed by EPR and electrochemical methods. Cyclic voltammetry of the heme-binding domain of human cytochrome b5 (HLMWb5) and its site-directed mutants was conducted using a gold electrode pre-treated with β-mercarptopropionic acid by inclusion of positively-charged poly-L-lysine. On the other hand, static midpoint potentials were measured under a similar condition.ResultsTitration of HLMWb5 with poly-L-lysine indicated that half-wave potential up-shifted to -19.5 mV when the concentration reached to form a complex. On the other hand, midpoint potentials of -3.2 and +16.5 mV were obtained for HLMWb5 in the absence and presence of poly-L-lysine, respectively, by a spectroscopic electrochemical titration, suggesting that positive charges introduced by binding of poly-L-lysine around an exposed heme propionate resulted in a positive shift of the potential. Analyses on the five site-specific mutants showed a good correlation between the half-wave and the midpoint potentials, in which the former were 16~32 mV more negative than the latter, suggesting that both binding of poly-L-lysine and hydrophobicity around the heme moiety regulate the overall redox potentials.ConclusionsPresent study showed that simultaneous measurements of the midpoint and the half-wave potentials could be a good evaluating methodology for the analyses of static and dynamic redox properties of various hemoproteins including cytochrome b5. The potentials might be modulated by a gross conformational change in the tertiary structure, by a slight change in the local structure, or by a change in the hydrophobicity around the heme moiety as found for the interaction with poly-L-lysine. Therefore, the system consisting of cytochrome b5 and its partner proteins or peptides might be a good paradigm for studying the biological electron transfer reactions.

NADH:Cytochrome b5 Reductase and Cytochrome b5 Can Act as Sole Electron Donors to Human Cytochrome P450 1A1-Mediated Oxidation and DNA Adduct Formation by Benzo[a]pyrene

“…We demonstrated that the reaction cycle of BaP oxidation catalyzed by human P450 1A1 can proceed by ways that differ from the generally accepted mechanism, where the first reduction of P450 is considered to be catalyzed by POR without cytochrome b 5 . 20 , 48 − 52 Considering the redox potentials of cytochrome b 5 (+20 mV) 53 , 54 and ferric substrate-bound P450 (−237 mV), 27 , 53 it is thermodynamically impossible for cytochrome b 5 to provide the first electron in the P450 catalytic cycle. 55 Given that the redox potential of oxyferrous P450 is also approximately +20 mV, it is feasible that cytochrome b 5 can supply the second electron into the catalytic cycle.…”

Miniature direct electron transfer based sulphite/oxygen enzymatic fuel cells

“…This value is ca. 0.1 V higher compared to the redox potential of b5-type cytochromes (Aono et al, 2010;Rivera et al, 1998), but it is in good agreement with the redox potential of hSOx (Johnson-Winters et al, 2010;Sezer et al, 2010). From the non-turnover reduction peak current (I p ) at a given scan rate v the surface coverage Γ of electrochemically active hSOx was calculated using Γ = I RT v F A 4 / n p 2 2 , where n is the number of electrons transferred, R the gas constant, T the temperature and F is the Faraday constant.…”

Accommodating a Nonconservative Internal Mutation by Water-Mediated Hydrogen Bonding between β-Sheet Strands: A Comparison of Human and Rat Type B (Mitochondrial) Cytochrome b5

“…58 Regardless of its source, we are confident that the redox potentials determined in this study for bCYB5A, rCYB5B and hCYB5B are valid for the stated conditions. Support for this conjecture is provided by the fact that cyclic voltammetry data for bCYB5A and rCYB5B performed in the presence of varying concentrations of divalent metal ions revealed a constant ~70 mV redox potential difference.…”

“…Type A cytochromes b 5 have been found to exhibit similar redox potentials (0 ± 10 mV; all reported potentials vs. SHE), 58–61 consistent with their highly conserved amino acid sequences. Reported redox potentials for rCYB5B (−102 mV) 22 and hCYB5B (−40 mV) 28 are more negative, suggesting a need for a stronger electron delivery driving force.…”