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 (ΔΔG(f)° = 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 (≈−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) × 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 (≈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) × 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 ≈4 ms [ΔG(f)° = −33(3) kJ mol(−1)] at zero denaturant.