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 histidineligated 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 g80%, which is consistent with the ultrafast photolysis time constant (e40 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 (τ e ) and large Soret-state electronic damping factor (Γ e ) 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. † Part of the special issue "Thomas Spiro Festschrift".

Effect of Adiabaticity on Electron Dynamics in Zinc Myoglobin

“…Others have indeed shown that related heme proteins such as cytochrome c undergo complex internal conversion dynamics. 43,44 One may also need to consider the effect of ligand dissociation and recombination of the proximal histidine sidechain. 45,46 A topic that the classical MD simulation used in this study was not capable of reproducing but an area worth investigating, in future work, with a coupled potential QMMM/ MD approach in which the formation and subsequent breaking of the N-Zn proximal histidine bond on a femtosecond time scale could be implicitly included.…”

Coherence Spectroscopy Investigations of the Low-Frequency Vibrations of Heme: Effects of Protein-Specific Perturbations

“…21,37 The "detuned" or "dispersed" detection scheme, on the other hand, leads to stronger relative enhancement of the higher frequency regions of the coherent signal. 21,[37][38][39] In this latter case, a photomultiplier tube (PMT) coupled to a monochromator was employed for detection, resulting in increased amplitude and superior detection of the higher frequency components of the third order polarization signal. This occurs because the induced polarization oscillates at the optical carrier frequency ± the coherent vibrational frequencies.…”

“…37,39,41 A more likely explanation is related to the lowered heme symmetry that results from specific protein-induced geometric distortions. The symmetry of the heme controls which specific modes are allowed in the coherence spectra.…”

Imaging chromophores with undetectable fluorescence by stimulated emission microscopy

“…Here we perform ex vivo stimulated emission imaging of the well-developed vascular network from a nude mouse ear, by exciting the Soret band of hemoglobin through efficient two-photon absorption 28 and subsequently stimulating the emission from its Q band which has longer excited state lifetime than Soret band 29 . As shown in Fig.…”