- 作者: Tomomi Aono; Yoichi Sakamoto; Masahiro Miura; Fusako Takeuchi; Hiroshi Hori; Motonari Tsubaki
- 作者服務機構: Department of Chemistry, Graduate School of Science, Kobe University, Kobe, Japan
- 中文摘要: --
- 英文摘要:
Background:Cytochrome 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.
Methods: Effects 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.
Results: Titration 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. 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 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. - 中文關鍵字: --
- 英文關鍵字: --