Abstract
We improved an already existing cytochrome c expression system to a reliable, tightly controllable one to achieve a higher expression yield for single cysteine mutants of horse cytochrome c. The protein is heterologously overexpressed in E. coli together with the maturation coordinating enzyme heme lyase from yeast. Various plasmid constructs and host strains were tested for protein expression yield and routinely around 35 mg/L yield was achieved, which is a good result for a post-translationally modified enzyme. The purpose of producing cysteine mutants is to position accessible cysteine residues on the surface of cytochrome c which can be labeled with a photoactive redox dye, 8-thiouredopyrene-1,3,6-trisul-fonate, TUPS. TUPS labeled proteins have been used for intramolecular and intermolecular electron transfer measurements. Here, we initiate the photoreduction of cytochrome c oxidase, the natural electron acceptor partner of cytochrome c by an appropriate cytochrome c mutant labeled with TUPS. The electron transfer from cytochrome c to the first cytochrome oxidase redox cofactor, copper A, is shown to be very fast.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. (eds) (1996) Current protocols in molecular biology. In: Current protocol series. Chanda, V. B. (serial ed.), Wiley, New York.
Dolgikh, D. A., Latypov, R. E., Abdullaev, Z. Kh., Kolon, V., Roder, H., Kirpichnikov, M. P. (1998) Expression of mutant genes for horse cytochrome c in Escherichia coli. Russian J. Bioorg. Chem. 24, 756.
Faxen, K., Gilderson, G., Adelroth, R., Brzezinski, P. (2005) A mechanistic principle for proton pumping by cytochrome c oxidase. Nature 437, 286–289.
Guzman, L. M., Belin, D., Carson, M. J., Beckwith, J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the Arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., Pease, L. R. (1989) Site directed mutagenesis by overlap extension using the polymerase chain-reaction. Gene 77, 51–59.
Hosier, J. P., Ferguson-Miller, S., Mills, D. A. (2006) Energy transduction: Proton transfer through the respiratory complexes. Annu. Rev. Biochem. 75, 165–187.
Inoue, H., Nojima, H., Okoyama, H. (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23–28.
Kotlyar, A. B., Borovok, N., Hazani, M. (1997) Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate cytochrome c derivatives. Biochemistry 36, 15828–15833.
Kranz, R., Lill, R., Goldman, B., Bonnard, G., Merchant, S. (1998) Molecular mechanisms of cytochrome c biogenesis: three distinct systems. Mol. Microbiol. 29, 383–396.
Pollock, W. B. R., Rosell, F. I, Twitchett, M. B., Dumont, M. E., Mauk, A. G. (1998) Bacterial expression of a mitochondrial cytochrome c. Trimethylation of Lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition. Biochemistry 37, 6124–6131.
Rumbley, J. N., Hoang, L., Englander, S. W. (2002) Recombinant equine cytochrome c in E. coli: high level expression, characterization, and folding and assembly mutants. Biochemistry 41, 13894–13901.
Sambrook, J., Maniatis, T., Fritsch, E. F. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Schleif R. (2000) Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics 16, 559–565.
Solem, C., Jensen, P. R. (2002) Modulation of gene expression made easy. Appl. Envir Microbiol. 68, 2397–2403.
Szundi, I., Cappuccio, J. A., Borovok, N., Kotlyar, A. B., Einarsdottir, O. (2001) Photoinduced electron transfer in the cytochrome c/ cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c. optical multichannel detection. Biochemistry 40, 2186–2193.
Tenger, K., Khoroshyy, P., Leitgeb, B., Rakhely, G., Borovok, N., Kotlyar, A., Dolgikh, D. A., Zimanyi, L. (2005) Complex kinetics of the electron transfer between the photoactive redox label TUPS and the heme of cytochrome c. J. Chem. Inf. Model. 45, 1520–1526.
Vaillancourt, P. E. (2002) E. coli Gene Expression Protocols. Applied Molecular Evolution. San Diego, CA, Humana Press.
Yoshikawa, S., Muramoto, K., Shinzawa-Itoh, K., Aoyama, H., Tsukihara, T., Shimokata, K., Katayama, Y., Shimada, H. (2006) Proton pumping mechanism of bovine heart cytochrome c oxidase. Biochim. Biophys. Acta 1757, 1110–1116.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
About this article
Cite this article
Tenger, K., Khoroshyy, P., Kovács, K.L. et al. Improved System For Heterologous Expression of Cytochrome C Mutants in Escherichia Coli. BIOLOGIA FUTURA 58 (Suppl 1), 23–35 (2007). https://doi.org/10.1556/ABiol.58.2007.Suppl.3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1556/ABiol.58.2007.Suppl.3