Evidence for formation of two thioether bonds to link heme to apocytochrome c by partially purified cytochrome c synthetase.

Cytochrome c synthetase has been solubilized from yeast mitochondria using Triton X-100 and fractionated with ammonium sulfate. Use of this partially purified enzyme has permitted us to isolate a quantity of iso-1-cytochrome c formed from 125I-labeled apocytochrome c and hemin in the presence of a NADPH-generating system. Visible absorption spectra (pH 8.0 or 5.0) including alpha, beta, and Soret bands and their molar absorption coefficients of this enzymatically synthesized cytochrome c in the oxidized and reduced states are the same, within experimental error, as those of native cytochrome c. Pyridine ferrohemochrome (pH 13) of the synthesized species also exhibits the same alpha and beta bands as those of iso-l-cytochrome c and similar to those reported for heme peptides of cytochrome c. If only one or no thioether bond were formed between the two vinyl side groups of heme and the cysteine residues of apocytochrome c, all these alpha and beta bands would have shifted to red (Pettigrew, G. W., Leaver, J. L., Meyer, T. E., and Ryle, T. E. (1975) Biochem J. 147, 291-302). Thus, two thioether bonds appear to be formed to link heme to apocytochrome c by cytochrome c synthetase, completing information of the three-dimensional structure of cytochrome c.