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Cytochrome c' folding triggered by electron transfer: Fast and slow formation of four-helix bundles

Lee, Jennifer C. and Gray, Harry B. and Winkler, Jay R. (2001) Cytochrome c' folding triggered by electron transfer: Fast and slow formation of four-helix bundles. Proceedings of the National Academy of Sciences of the United States of America, 98 (14). pp. 7760-7764. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:LEEpnas01

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Abstract

Reduced (Fe-II) Rhodopseudomonas palustris cytochrome c' (Cyt c') is more stable toward unfolding ([GuHCl](1/2) = 2.9(1) M) than the oxidized (Fe-III protein ([GuHCl](1/2) = 1.9(1) M). The difference in folding free energies (Delta DeltaG(f)degrees = 70 meV) is less than half of the difference in reduction potentials of the folded protein (100 mV vs. NHE) and a free heme in aqueous solution (approximate to -150 mV). The spectroscopic features of unfolded Fe-II-Cyt c' indicate a low-spin heme that is axially coordinated to methionine sulfur (Met-15 or Met-25). Time-resolved absorption measurements after CO photodissociation from unfolded Fe-II(CO)-Cyt c' confirm that methionine can bind to the ferroheme on the microsecond time scale [k(obs) = 5(2) x 10(4) s(-1)]. Protein folding was initiated by photoreduction (two-photon laser excitation of NADH) of unfolded Fe-III-Cyt c' ([GuHCl] = 2.02-2.54 M). Folding kinetics monitored by heme absorption span a wide time range and are highly heterogeneous; there are fast-folding (approximate to 10(3) s(-1)), intermediate-folding (10(2)-10(1) s(-1)), and slow-folding (10(-1) s(-1)) populations, with the last two likely containing methionine-ligated (Met-15 or Met-25) ferrohemes. Kinetics after photoreduction of unfolded Fe-III-Cyt c' in the presence of CO are attributable to CO binding [1.4(6) x 10(3) s(-1)] and Fe-II(CO)-Cyt c' folding [2.8(9) s(-1)] processes; stopped-flow triggered folding of Fe-III-Cyt c' (which does not contain a protein-derived sixth ligand) is adequately described by a single kinetics phase with an estimated folding time constant of approximate to 4 ms [DeltaG(f)degrees = -33(3) kJ mol(-1)] at zero denaturant.


Item Type:Article
Additional Information:Copyright © 2001 by the National Academy of Sciences. Contributed by Harry B. Gray, May 11, 2001. We thank Michael Cusanovich and Terry Meyer (University of Arizona) for assistance with protein isolation and purification, Brian Crane for help with protein structure modeling, and Akif Tezcan for discussions. J.C.L. thanks the Ralph M. Parsons Foundation for a graduate fellowship. This work was supported by National Science Foundation Grant MCB-9974477 and by the Arnold and Mabel Beckman Foundation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Subject Keywords:NUCLEAR-MAGNETIC-RESONANCE, RHODOPSEUDOMONAS-PALUSTRIS, BACTERIAL CYTOCHROMES-C', FERRICYTOCHROME C', CONTACT FORMATION, ENERGY LANDSCAPE, ESCHERICHIA-COLI, TRANSITION-STATE, 4-HELIX BUNDLE, PROTEIN
Record Number:CaltechAUTHORS:LEEpnas01
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:LEEpnas01
Alternative URL:http://dx.doi.org/10.1073/pnas.141235198
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:983
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:21 Nov 2005
Last Modified:26 Dec 2012 08:42

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