The (t approximately 0) photodissociation quantum yields (Y(0)) of MbNO and MbO(2) are measured to be 50 +/- 5 and 28 +/- 6%, respectively, using MbCO (Y(0) = 100%) as a reference. When photolysis does not take place, we find that a significant portion of the photon energy contributes to heating of the residual six-coordinate heme (MbNO and MbO(2)). The time constant for vibrational relaxation of the six-coordinate ligand-bound heme is found to be close to 1 ps for both samples. The MbO(2) sample also shows a approximately 4-ps optical response that is assigned to a rapid phase (25-30% amplitude) of O(2) geminate rebinding. We observe no additional geminate recombination in the MbO(2) sample out to 120 ps. In contrast, the MbNO sample displays significant geminate recombination over the first 120 ps, which can be adequately fit with two exponentials whose amplitudes and time constants appear to depend weakly on the pump wavelength. This more complex kinetic behavior conceivably arises due to heating of the photodissociated heme and its effect on the geminate recombination as the system cools. Overall, the data are consistent with a hypothesis that distortions along the iron-ligand bending coordinate play a key role in the photodissociation process. The transient formation of an unphotolyzable FeO(2) side-on binding geometry is suggested to be responsible for the lowered quantum yield of MbO(2) relative to MbNO.

The rebinding kinetics of NO to the heme iron of myoglobin (Mb) is investigated as a function of temperature. Below 200K, the transition state enthalpy barrier associated with the fastest (~10ps) recombination phase is found to be zero, while a slower geminate phase (~200ps) reveals a small enthalpic barrier (~ 3 ± 1 kJ/mol). Both of the kinetic rates slow down slightly in the myoglobin (Mb) samples above 200K, suggesting that a small amount of protein relaxation takes place above the solvent glass transition. When the temperature dependence of the NO recombination in Mb is studied under conditions where the distal pocket is mutated (e.g., V68W), the rebinding kinetics lack the slow phase. This is consistent with a mechanism where the slower (~200ps) kinetic phase involves transitions of the NO ligand into the distal heme pocket from a more distant site (e.g., in or near the Xe4 cavity). Comparison of the temperature dependent NO rebinding kinetics of native Mb with that of the bare heme (PPIX) in glycerol reveals that the fast (enthalpically barrierless) NO rebinding process observed below 200K is independent of the presence or absence of the proximal histidine ligand. In contrast, the slowing of the kinetic rates above 200K in MbNO disappears in the absence of the protein. Generally, the data indicate that, in contrast to CO, the NO ligand binds to the heme iron through a "harpoon" mechanism where the heme iron out-of-plane conformation presents a negligible enthalpic barrier to NO rebinding. These observations strongly support a previous analysis (J. Am. Chem. Soc. 1988, 110, 6656) that primarily attributes the low temperature stretched exponential rebinding of MbCO to a quenched distribution of heme geometries. A simple model is presented for MbNO rebinding that explains a variety of experiments, including the dependence of the kinetic amplitudes on the pump photon energy.

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".

The technique of femtosecond coherence spectroscopy (FCS) is applied to the heme protein myoglobin. Photostable samples of deoxy myoglobin (Mb) and photochemically active samples of the nitric oxide adduct (MbNO) are investigated. The pump-induced change in the probe transmittance for both samples displays coherent oscillations that, when transformed into the frequency domain, are in agreement with the resonance Raman spectrum of deoxy Mb. This indicates that the coherences associated with the photoreactive sample (MbNO) arise from the rapidly changing forces appearing in the crossing region(s) between the reactant and product state potential energy surfaces. The relative phase and amplitude of the Fe-His vibration, associated with the sole covalent linkage between the heme and the protein, are analyzed as a function of sample state and pump/probe carrier frequency. The dependence of the phase on carrier frequency is found to be significantly different for the "field driven" coherence in Mb and the "reaction driven" coherence in MbNO. In MbNO we observe a dip in amplitude and a phase flip near 439 nm for the Fe-His mode, whereas in deoxy Mb we observe a nearly constant phase and amplitude for this mode across the Soret absorption band. These observations are shown to be in good agreement with a simple theoretical model of the pump-probe experiment. Finally, we present recent observations of strong low-frequency oscillations, occurring near 40 cm -1 in both species and near 80 cm -1 for MbNO.