Direct electrochemical analyses of human cytochromes b(5) with a mutated heme pocket showed a good correlation between their midpoint and half wave potentials

作者:Aono Tomomi; Sakamoto Yoichi; Miura Masahiro; Takeuchi Fusako; Hori Hiroshi; Tsubaki Motonari*
来源:Journal of Biomedical Science, 2010, 17(1): 90.
DOI:10.1186/1423-0127-17-90

摘要

Background: Cytochrome b(5) 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 b(5) span a very wide range of similar to 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. Methods: Effects of mutations at conserved hydrophobic amino acid residues consisting of the heme pocket of cytochrome b(5) were analyzed by EPR and electrochemical methods. Cyclic voltammetry of the heme-binding domain of human cytochrome b(5) (HLMWb(5)) and its site-directed mutants was conducted using a gold electrode pre-treated with beta-mercarptopropionic acid by inclusion of positively-charged poly-L-lysine. On the other hand, static midpoint potentials were measured under a similar condition. Results: Titration of HLMWb(5) 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 HLMWb(5) 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 similar to 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. Conclusions: Present 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 b(5). 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 b(5) and its partner proteins or peptides might be a good paradigm for studying the biological electron transfer reactions.

  • 出版日期2010-12-4