Investigations of photophysical and photochemical processes in heme proteins through ultrafast laser spectroscopy and low-temperature absorption spectroscopy

Photon-induced physical and chemical processes in heme proteins have been extensively studied through femtosecond coherence spectroscopy, transient absorption spectroscopy and low temperature spectroscopy.;Near-infrared absorption spectra of ferrous P450cam have been obtained at both room temperature and cryogenic temperature. The charge-transfer [e g(dxz) → eg(pi*)] transition at ∼1100 nm (Band I) has been found to be a conformational sensitive indicator of the heme-pocket geometry in P450cam. Both temperature and ligand rebinding affect the line width and position of this transition. We conclude that the overall line shape of Band I is derived from different conformational substrates which mainly result from iron doming and proximal cysteine tilting within each protein.;The technique of femtosecond coherence spectroscopy (FCS) is applied to simple transparent organic fluids (chloroform and fenchone) and light absorbing heme proteins (myoglobin derivatives). This study demonstrates that time-domain coherence measurements are in good agreement with standard frequency domain Raman and resonance Raman scattering experiments. We also demonstrate the unique capability of FCS to detect the low frequency nuclear motions driven by the pumping fields and by the electronic state changes associated with photoinduced chemical reactions. The latter response can not be probed using traditional Raman spectroscopy. In all studies of myoglobin derivatives, cytochrome c, and microperoxidase, we observed a low frequency mode near 40 cm -1. We also observed a sequence of low frequency modes near 80 cm-1, 120 cm-1 and 160 cm -1 that could be an indication of an overtone sequence or anharmonic potential surface.;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 cm-1, ∼80 cm -1 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 (tau e) and large Soret state electronic damping factor (Gammae) 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.