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-II I protein ([GuHCl](1/2) = 1.9(1) M), The difference in folding free energie s (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 o f unfolded Fe-II-Cyt c ' indicate a low-spin heme that is axially coordinat ed to methionine sulfur (Met-15 or Met-25), Time-resolved absorption measur ements after CO photodissociation from unfolded Fe-II(CO)-Cyt c ' confirm t hat methionine can bind to the ferroheme on the microsecond time scale [k(o bs) = 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 ti me 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 un folded 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)] process es; stopped-flow triggered folding of Fe-III-Cyt c ' (which does not contai n a protein-derived sixth ligand) is adequately described by a single kinet ics phase with an estimated folding time constant of approximate to4 ms [De ltaG(f)degrees = -33(3) kJ mol(-1)] at zero denaturant.