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

Topologically homologous four-helix-bundle heme proteins exhibit striking diversity in their refolding kinetics. Cytochrome b562 has been reported to fold on a sub-millisecond time scale, whereas cytochrome c' refolding requires 10 s or more to complete. Heme dissociation in cytochrome b562 interferes with studies of folding kinetics, so a variant of cytochrome b562 (cytochrome c-b562) 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-b562, along with those of Fe(II)-cytochrome c556, have been examined by using time-resolved absorption spectroscopy. Coordination of S(Met) to Fe(II) occurs within 10 mus after reduction of the denatured Fe(III)-cytochromes, and shortly thereafter (100 micros) the heme spectra are indistinguishable from those of the folded proteins. Under denaturing conditions, carbon monoxide binds to the Fe(II)-hemes in approximately 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.