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

The low-frequency mode activity of metalloporphyrins has been studied for iron porphine-halides (Fe(P)(X), X = Cl, Br) and nitrophorin 4 (NP4) using femtosecond coherence spectroscopy (FCS) in combination with polarized resonance Raman spectroscopy and density functional theory (DFT). It is confirmed that the mode symmetry selection rules for FCS are the same as for Raman scattering and that both Franck-Condon and Jahn-Teller mode activities are observed for Fe(P)(X) under Soret resonance conditions. The DFT-calculated low-frequency (20-400 cm -1 ) modes, and their frequency shifts upon halide substitution, are in good agreement with experimental Raman and coherence data, so that mode assignments can be made. The doming mode is located at ~80 cm -1 for Fe(P)(Cl) and at ~60 cm -1 for Fe(P)(Br). NP4 is also studied with coherence techniques, and the NO-bound species of ferric and ferrous NP4 display a mode at ~30-40 cm -1 that is associated with transient heme doming motion following NO photolysis. The coherence spectra of three ferric derivatives of NP4 with different degrees of heme ruffling distortion are also investigated. We find a mode at ~60 cm -1 whose relative intensity in the coherence spectra depends quadratically on the magnitude of the ruffling distortion. To quantitatively account for this correlation, a new "distortion-induced" Raman enhancement mechanism is presented. This mechanism is unique to low-frequency "soft modes" of the molecular framework that can be distorted by environmental forces. These results demonstrate the potential of FCS as a sensitive probe of dynamic and functionally important nonplanar heme vibrational excitations that are induced by the protein environmental forces or by the chemical reactions in the aqueous phase.

Significance To probe the effect of heme ruffling on electron transport, we studied three cytochromes that display wide variation in the heme ruffling distortion. Ruffling is characterized by a low-frequency heme mode in the region 45–60 cm −1 and by a photoreduction cross-section that displays strong variation as a function of the magnitude of the distortion. Given the similarity in the distance between the heme and the nearest aromatic amino acid for all three proteins, the order-of-magnitude changes in photoreduction rate demonstrate that the ruffling coordinate can serve as a control mechanism for electron transport in heme proteins. Major differences in heme ruffling are noted for cytochrome c when bound to the mitochondrial membrane compared with its solution structure.

Nitrophorin 4 (NP4) is a heme protein that stores and delivers nitric oxide (NO) through pH sensitive conformational change. This protein uses the ferric state of a highly ruffled heme to bind NO tightly at low pH and release it at high pH. In this work, the rebinding kinetics of NO and CO to NP4 are investigated as a function of iron oxidation state and the acidity of the environment. The geminate recombination process of NO to ferrous NP4 at both pH 5 and pH 7 is dominated by a single ~7 ps kinetic phase that we attribute to the rebinding of NO directly from the distal pocket. The lack of pH dependence explains in part why NP4 cannot use the ferrous state to fulfill its function. The kinetic response of ferric NP4NO shows two distinct phases. The relative geminate amplitude of the slower phase increases dramatically as the pH is raised from 5 to 8. We assign the fast phase of NO rebinding to a conformation of the ferric protein with a closed hydrophobic pocket. The slow phase is assigned to the protein in an open conformation with a more hydrophilic heme pocket environment. Analysis of the ultrafast kinetics finds the equilibrium off-rate of NO to be proportional to the open state population as well as the pH-dependent amplitude of escape from the open pocket. When both factors are considered, the off-rate increases by more than an order of magnitude as the pH changes from 5 to 8. The recombination of CO to ferrous NP4 is observed to have a large non-exponential geminate amplitude with rebinding timescales of ~10 −11 -10 −9 s at pH 5 and ~10 −10 -10 −8 s at pH 7. The nonexponential CO rebinding kinetics at both pH 5 and pH 7 are accounted for using a simple model that has proven effective for understanding CO binding in a variety of other heme systems.