Abstract
Studies on horse heart cytochrome c polymers were carried out by stopped-flow and photolysis techniques, to investigate the properties of the CO complex and the kinetics of electron transfer, mainly of the dimeric and tetrameric forms. CO binding, which does not occur with native monomers, proceeds at both pH7.0 and pH9.6, and appears to follow complex kinetics: an initial phase is observed, which is CO-concentration-dependent, followed by a very slow monomolecular phase (k~2×10−3s−1 at pH7) before establishment of equilibrium. Photodissociation of the CO complex has a very low quantum yield, probably less than 0.1. Static titration data of the dimer gave an `n' value of 0.4. These data strongly suggest heterogeneity of the population of binding sites, and have been interpreted in terms of the existence of different structures, probably owing to the non-unique type of binding of monomers during polymerization. Polymers of cytochrome c carboxymethylated on the methionine residue normally ligated to iron show simple CO recombination kinetics after photolytic removal (kon=1.5×106 m−1·s−1 at pH6). We therefore suggest that, for native cytochrome c, polymerization has an effect on the lability of the haem crevice, rendering the iron available for binding ligands, without, however, forming the structure of a truly open crevice. Electron transfer is, on the other hand, a simple process, and no gross differences are observed between monomer and polymers. A simple model, taking into account all these data, is suggested.
Full text
PDF





Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. doi: 10.1042/bj0910222. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gibson Q. H., Milnes L. Apparatus for rapid and sensitive spectrophotometry. Biochem J. 1964 Apr;91(1):161–171. doi: 10.1042/bj0910161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greenwood C., Gibson Q. H. The reaction of reduced cytochrome C oxidase with oxygen. J Biol Chem. 1967 Apr 25;242(8):1782–1787. [PubMed] [Google Scholar]
- Greenwood C., Palmer G. Evidence for the existence of two functionally distinct forms cytochrome c manomer at alkaline pH. J Biol Chem. 1965 Sep;240(9):3660–3663. [PubMed] [Google Scholar]
- Kaminsky L. S., Burger P. E., Davison A. J., Helfet D. Carbon monoxide as a probe for conformation changes of ferrocytochrome c. Biochemistry. 1972 Sep 26;11(20):3702–3706. doi: 10.1021/bi00770a007. [DOI] [PubMed] [Google Scholar]
- Lambeth D. O., Palmer G. The kinetics and mechanism of reduction of electron transfer proteins and other compounds of biological interest by dithionite. J Biol Chem. 1973 Sep 10;248(17):6095–6103. [PubMed] [Google Scholar]
- MARGOLIASH E., FROHWIRT N., WIENER E. A study of the cytochrome c haemochromogen. Biochem J. 1959 Mar;71(3):559–570. doi: 10.1042/bj0710559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MARGOLIASH E., LUSTGARTEN J. Interconversion of horse heart cytochrome C monomer and polymers. J Biol Chem. 1962 Nov;237:3397–3405. [PubMed] [Google Scholar]
- Margoliash E., Schejter A. Cytochrome c. Adv Protein Chem. 1966;21:113–286. doi: 10.1016/s0065-3233(08)60128-x. [DOI] [PubMed] [Google Scholar]
- SCHEJTER A., GEORGE P. THE 695-MMM. BAND OF FERRICYTOCHROME C AND ITS RELATIONSHIP TO PROTEIN CONFORMATION. Biochemistry. 1964 Aug;3:1045–1049. doi: 10.1021/bi00896a006. [DOI] [PubMed] [Google Scholar]
- SCHEJTER A., GLAUSER S. C., GEORGE P., MARGOLIASH E. SPECTRA OF CYTOCHROME C MONOMER AND POLYMERS. Biochim Biophys Acta. 1963 Aug 6;73:641–643. doi: 10.1016/0006-3002(63)90334-2. [DOI] [PubMed] [Google Scholar]
- Schejter A., Aviram I. The effects of alkylation of methionyl residues on the properties of horse cytochrome c. J Biol Chem. 1970 Apr 10;245(7):1552–1557. [PubMed] [Google Scholar]
- Shur-Perek T., Avi-Dor Y. The effect of cytochrome c and its 'dimer' on electron transfer and energy transformation. Biochem J. 1972 Feb;126(3):709–716. doi: 10.1042/bj1260709. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson M. T., Brunori M., Bonaventura J., Bonaventura C. Effect of steady illumination on the binding of carbon monoxide by carboxymethylated cytochrome c. Biochem J. 1973 Apr;131(4):863–865. doi: 10.1042/bj1310863. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson M. T., Brunori M., Rotilio G. C., Antonini E. Properties of modified cytochromes. II. Ligand binding to reduced carboxymethyl cytochrome c. J Biol Chem. 1973 Dec 10;248(23):8162–8169. [PubMed] [Google Scholar]
- Wilson M. T., Greenwood C. Studies on ferricytochrome c. 2. A correlation between reducibility and the possession of the 695mm absorption band of ferricytochrome c. Eur J Biochem. 1971 Sep 13;22(1):11–18. doi: 10.1111/j.1432-1033.1971.tb01508.x. [DOI] [PubMed] [Google Scholar]

