The facile replacement of heme c in cytochromes c with non-natural prosthetic groups has been difficult to achieve due to two thioether linkages between cysteine residues and the heme. Fee et al. demonstrated that cytochrome c(552) from Thermus thermophilus, overproduced in the cytosol of E. coli, has a covalent linkage cleavable by heat between the heme and Cys11, as well as possessing the thioether linkage with Cys14 [Fee, J. A. (2004) Biochemistry 43, 12162-12176]. Prompted by this result, we prepared a C14A mutant, anticipating that the heme species in the mutant was bound to the polypeptide solely through the thermally cleavable linkage; therefore, the removal of the heme would be feasible after heating the protein. Contrary to this expectation, C14A immediately after purification (as-purified C14A) possessed no covalent linkage. An attempt to extract the heme using a conventional acid-butanone method was unsuccessful due to rapid linkage formation between the heme and polypeptide. Spectroscopic analyses suggested that the as-purified C14A possessed a heme b derivative where one of two peripheral vinyl groups had been replaced with a group containing a reactive carbonyl. A reaction of the as-purified C14A with [BH(3)CN](-) blocked the linkage formation on the carbonyl group, allowing a quantitative yield of heme-free apo-C14A. Reconstitution of apo-C14A was achieved with ferric and ferrous heme b and zinc protoporphyrin. All reconstituted C14As showed spontaneous covalent linkage formation. We propose that C14A is a potential source for the facile production of an artificial cytochrome c, containing a non-natural prosthetic group.

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