title: Fast coordination changes in cytochrome c do not necessarily imply folding.
abstract: Folding of globular proteins occurs with rates that range from microseconds to minutes; consequently, it has been necessary to develop new strategies to follow the faster processes that exceed stopped-flow capabilities. Rapid photochemical methods have been employed to study the rate of folding of reduced cytochrome c. In this protein, the iron of the covalently bound heme binds a His and a Met, proximal and distal. Unfolding by guanidine or urea weakens the Fe-Met bond, and the reduced unfolded cytochrome c easily binds CO and other heme ligands, which would react slowly or not at all with the native protein. Therefore in the presence of CO, reduced cytochrome c unfolds at lower denaturant concentrations than in the absence of this ligand, and rapid photochemical removal of CO from unfolded cytochrome c, is expected to trigger at least an incomplete refolding. This approach is complicated by the breakage of the proximal His-Fe bond that may occur as a consequence of CO photodissociation in the unfolded cytochrome c because of the so-called base elimination mechanism. Rebinding of CO to the four-coordinate heme yields kinetic intermediates unrelated to folding. Our hypothesis is supported by parallel observations carried out with protoheme and microperoxidase.
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti e Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Dipartimento di Scienze Biochimiche Alessandro Rossi-Fanelli, Università di Roma La Sapienza, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Arcovito
authors.forename: S
authors.initials: S
authors.lastname: Gianni
authors.forename: M
authors.initials: M
authors.lastname: Brunori
authors.forename: C
authors.initials: C
authors.lastname: Travaglini-Allocatelli
authors.forename: A
authors.initials: A
authors.lastname: Bellelli
mesh_terms: D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D007700:Kinetics; D017510:Protein Folding
chemical_list: D003574:Cytochrome c Group; D002248:Carbon Monoxide