We present temperature-dependent kinetic measurements of ultrafast diatomic ligand binding to the ''bare'' protoheme (L 1-FePPIX-L 2, where L1 ‫؍‬ H2O or 2-methyl imidazole and L2 ‫؍‬ CO or NO). We found that the binding of CO is temperature-dependent and nonexponential over many decades in time, whereas the binding of NO is exponential and temperature-independent. The nonexponential nature of CO binding to protoheme, as well as its relaxation above the solvent glass transition, mimics the kinetics of CO binding to myoglobin (Mb) but on faster time scales. This demonstrates that the nonexponential kinetic response observed for Mb is not necessarily due to the presence of protein conformational substates but rather is an inherent property of the solvated heme. The nonexponential kinetic data were analyzed by using a linear coupling model with a distribution of enthalpic barriers that fluctuate on slower time scales than the heme-CO recombination time. Below the solvent glass transition (T g Ϸ 180 K), the average enthalpic rebinding barrier for H 2O-PPIX-CO was found to be Ϸ1 kJ/mol. Above T g, the barrier relaxes and is Ϸ6 kJ/mol at 290 K. Values for the first two moments of the heme doming coordinate distribution extracted from the kinetic data suggest significant anharmonicity above T g. In contrast to Mb, the protoheme shows no indication of the presence of ''distal'' enthalpic barriers. Moreover, the wide range of Arrhenius prefactors (10 9 to 10 11 s ؊1 ) observed for CO binding to heme under differing conditions suggests that entropic barriers may be an important source of control in this class of biochemical reactions.heme proteins

We demonstrate novel optical scanning and detection instrumentation that is particularly useful for the interrogation of stationary cryogenic samples in pump-probe spectroscopy. The technique uses a spinning lens to scan multiple laser beams over a stationary sample while maintaining the focal properties of the beams. This significantly lengthens the time window for the sample reset to equilibrium and improves the photostability of stationary samples. In addition, we describe a signal processing methodology that discriminates against the strong background signal that can arise from leakage of the pump laser pulse train into the detector. These techniques are particularly useful in pump-probe studies of ultrafast processes in biological systems where sample deterioration, pump induced thermal lensing, and light scattering into the detection channel ͑e.g., induced by light scattering from a cryogenic matrix͒ are problematic. Generally, the optical scanning and detection instrumentation described here enable the study of a variety of biological systems, without the need for intricate spinning or flowing sample containers, making it possible to perform ultrafast pump-probe experiments on stationary samples such as a cryogenic matrix.