Early events in the folding of four-helix-bundle heme proteins

Abstract

Topologically homologous four-helix-bundle heme proteins exhibit striking diversity in their refolding kinetics. Cytochrome b(562) has been reported to fold on a submillisecond time scale, whereas cytochrome c' refolding requires 10 s or more to complete. Heme dissociation in cytochrome b(562) interferes with studies of folding kinetics, so a variant of cytochrome b(562) (cytochrome c-b(562)) with a covalent c-type linkage to the heme has been expressed in Escherichia coli. Early events in the electron transfer-triggered folding of Fe-II-cytochrome c-b(562), along with those of Fe-II-cytochrome c(556), have been examined by using time-resolved absorption spectroscopy. Coordination of S(Met) to Fe-II occurs within 10 mu(s) after reduction of the denatured Fe-III-cytochromes, and shortly thereafter (100 mu(s)) the heme spectra are indistinguishable from those of the folded proteins. Under denaturing conditions, carbon monoxide binds to the Fe-II-hemes in approximate to 15 ms. By contrast, CO binding cannot compete with refolding in the Fe-II-cytochromes, thereby confirming that the polypeptide encapsulates the heme in <10 ms. We suggest that Fe-S(Met) ligation facilitates refolding in these four-helix-bundle heme proteins by reducing the conformational freedom of the polypeptide chain.

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