76NISR RESEARCH GRANT
2004 Research Grant

Function of Thermostable Redox Proteins as Thermo-electric
Energy Converters
Yoshihiro SAMBONGI
Graduate School of Biosphere Science, Hiroshima University
Research aims
Purpose of this study is elucidation of molecular property of biological energy metabolism in an aspect of application science and technology. We will deal with a thermally stable redox protein, cytochrome c, and apply it touse as a thermo-electric energy converter. We have established basic technology in handling of various cytochromesc1). Here we try to understand functional mechanism of cytochrome c, and gain insight into a novel strategy in industrial energy production.
Methods
This study consists of three sub-projects.
(1) Structure, stability, and electron transfer reaction ofcytochromes c: Five cytochromes c from five Gram-negative bacteria, (I) Shewanella violacea (growing at 8°C), (II)Pseudomonas aeruginosa (37°C), (III) Hydrogenophilus
thermoluteolus (52°C), (IV) Hydrogenobacter thermophilus(72°C), and (V) Aquifex aeolicus (85°C), were used in thisstudy. These cytochromes c were tested for thermal andchemical denaturation using circular dichroic (CD) spectroscopy and for redox function derived from electrontransfer reaction using cyclic voltammetry (CV).
(2) Cytochrome c expression: We tested how the cytochromes c (I to V above) were able to be expressed in Escherichia coli efficiently used as a host organism. We examined gene structure to be expressed, and selected suitable E.
coli host strains.
(3) Temperature dependent redox potential: We measured temperature dependency of redox potential of the cytochromes c by CV. Furthermore, we searched for engineered cytochromes c that had larger difference in redox
poteintial during temperature change.
Results(1) Structure, stability, and electron transfer reaction ofcytochromes c: We found that the order of stability of cytochromes c from bacteria (II) to (V) was the same as thatof the growth temperature of original bacteria2). The stability of protein from (I) was the same as that of (II). This isbecause that the cytochrome c from (I) has a unique SS
bond, which contributes to the enhanced stability. Furthermore, the protein from (V) was extremely stable; it was notdenatured at 130°C, or with 7 M denaturant, guanidine hy-drochloride at pH 5.0.
We also measured redox potential at 25°C for the proteins from (II) and (IV). We were able to unify the free energy change during redox reaction and that during protein
denaturation3).
(2) Cytochrome c expression: The cytochrome c from(II) was expressed in E. coli, and the yield reached up to30 mg product per 1 liter cell culture. However, the othercytochromes c were not expressed when the intact genes
were introduced into E. coli. We found that these cytochromes c were expressed in E. coli when the genes werefused with the periplasmic targeting signal pepetide fromthe (II) protein2).
Furthermore, the expression of most cytochromes c wasdependent on Dsb proteins which were responsible forheme attachment in E. coli cells. Exceptionally, however,the protein from (V) was not dependent on the Dsb proteins, and the expression level was higher without the Dsb
proteins than with them4).
(3) Temperature dependent redox potential: We foundthat redox poteintials of all the proteins tested here werehigher at lower temperature than at higher temperature5).
These results indicate that the proteins can generate electricity with changing temperature. Furthermore, we foundthat a variant of (III) protein carrying single mutation
showed elevated stability and larger temperature dependency in redox potential.
Conclusion
The progress of this study is that we gain insight into (i)relationship between stability and redox poteintial in cytochromes c, (ii) maximization of cytochrome c expression,(iii) possibility in generating electric power using cytochromes c with temperature change. These findings may
be a base in developing thermo-electric energy converters
using electron-transferring proteins, cytochromes c which
are thermally stable.
References
1.

Sambongi, Y. et al., Cytochrome c from a thermophilic bacterium
has provided insights into the mechanism of protein maturation, folding, and stability. Eur. J. Biochem., 269, 3355–3361 (2002).
2. Oikawa, K. et al., Five amino acid residues responsible for the highstability of Hydrogenobacter thermophilus cytochrome c552: RECIPROCAL MUTATION ANALYSIS. J. Biol. Chem., 280, 5527–5532(2005).

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Uchiyama, S. et al., Complete thermal-unfolding profiles of oxidized
and reduced cytochromes c. J. Am. Chem. Soc., 126, 14684–14685(2004).
4) Kojima, N. et al., Unexpected and elevated production of Aquifex aeolicus cytochrome c555 in Escherichia coli cells lacking disulfide oxi-5)

doreductases. Biosci. Biotech. Biochem., 69, 1418–1421 (2005).
Takayama, S. J. et al., Control of the redox potential of
Pseudomonas aeruginosa cytochrome c551 through the Fe-Met coordination bond strength and pKa of a buried heme propionic acid sidechain. Biochemistry, 44, 5488–5494 (2005).