title: 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 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.
authors.affiliation: Beckman Institute, California Institute of Technology, Pasadena, CA 91125-7400, USA.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Faraone-Mennella
authors.forename: Harry B
authors.initials: HB
authors.lastname: Gray
authors.forename: Jay R
authors.initials: JR
authors.lastname: Winkler
mesh_terms: D002248:Carbon Monoxide / Q000378:metabolism; D002942:Circular Dichroism; D045303:Cytochromes b / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism*; D004926:Escherichia coli; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
chemical_list: D006418:Heme; D002248:Carbon Monoxide; D045304:Cytochromes c; D045303:Cytochromes b