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

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.