Femtosecond Multicolor Pump−Probe Spectroscopy of Ferrous Cytochrome c

Abstract

Ligand photolysis and subsequent electronic and structural relaxation, followed by ligand recombination, in ferrous cytochrome c (cyt c) have been studied using ultrafast laser spectroscopy. A broad-band white-light continuum, generated by amplified pulses from a Ti:sapphire laser, was used to monitor the transient absorption spectra of cyt c in the Soret and Q bands following 50-fs pulsed photoexcitation at 400 nm. The reconstructed photoproduct absorption spectrum is found to closely resemble that of a model pentacoordinate histidine-ligated complex, microperoxidase (MP-8), suggesting methionine photolysis. Vibrational modes at ∼40, ∼80, and ∼220 cm-1 are observed in femtosecond coherence spectroscopy (FCS) measurements, which also indicates photodissociation of the methionine ligand. The quantum yield of ligand photolysis is found to be ≥80%, which is consistent with the ultrafast photolysis time constant (≤40 fs) needed to induce coherent oscillations in the FCS measurements. The combination of high quantum yield and short time constant helps to resolve the longstanding question of the origin of the short lifetime (τe) and large Soret-state electronic damping factor (Γe) previously found in cytochrome c. We propose a simple multilevel model to describe the observed experimental data. The global analysis of the measured kinetics leads to a characterization of the major kinetic rates, including the 6.2-ps geminate rebinding of methionine to the heme iron.