Cytochrome c' folding triggered by electron transfer: fast and slow formation of four-helix bundles.
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 Delta G(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 ( approximately -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 ( approximately 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 approximately 4 ms [Delta G(f) degrees = -33(3) kJ mol(-1)] at zero denaturant.
Lee
J C
JC
Beckman Institute, MC 139-74, California Institute of Technology, Pasadena, CA 91125-7400, USA.
Gray
H B
HB
Winkler
J R
JR
D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D017510:Protein Folding; D012241:Rhodopseudomonas / Q000378:metabolism; D013329:Structure-Activity Relationship
D003574:Cytochrome c Group