Ultrafast CO Kinetics in Heme Proteins: Adiabatic Ligand Binding and Heavy Atom Tunneling

Abstract

We report on the ultrafast kinetics of CO rebinding to carbon monoxide oxidation activator protein (ChCooA) over a wide temperature range and make comparisons with the kinetics of CO and NO binding to protoheme (Fe protoporphyrin IX) and myoglobin (Mb). The CO binding to ChCooA is non-exponential over many decades in time at all temperatures studied, including room temperature. To describe this kinetic response we use a linear coupling model with a distribution of enthalpic rebinding barriers that is attributed primarily to protein-induced heterogeneity in the heme doming conformation (distributed barrier model, DBM). Above the solvent glass transition (Tg ~180K), CO rebinding kinetics displays an anti-Arrhenius behavior (i.e., the rate decreases as temperature increases) and this is ascribed to an evolution of the distribution toward increased heme doming and larger enthalpic barriers. Between Tg and ~60K, the non-exponential rebinding slows down as the temperature is lowered and the survival fraction follows the predictions expected for a quenched barrier distribution. However, below ~60K the rebinding kinetics do not continue to slow as predicted by the thermally activated DBM. A possible explanation for this behavior is explored that involves quantum mechanical tunneling of the iron atom along the heme doming coordinate. When the ultrafast CO rebinding kinetics of CooA are compared to the much slower CO rebinding in myoglobin, a two order-of-magnitude increase in the Arrhenius prefactor, from ~109 s−1 to ~1011 s−1, is revealed. A similar prefactor is found for NO binding to CooA, which is typical for NO rebinding to ferrous heme systems. Because kinetic studies of CO rebinding to protoheme also reveal prefactors near ~1011 s−1, we revisit the commonly held view that the CO binding reaction is non-adiabatic due to spin-forbidden (ΔS=2) selection rules. A non-adiabatic sequential mechanism, involving first order (ΔS=1) transitions, is considered as a possible explanation for ultrafast CO rebinding; however, the relative crossing energies of the relevant spin states leads us to conclude that the CO ligand binding reaction is adiabatic rather than non-adiabatic and that entropic factors, rather than spin-selection rules, are the cause of the reduced Arrhenius prefactor for CO binding in Mb and Hb.

A mechanism for CO regulation of ion channels

“…Multiphasic geminate rebinding behaviour is observed in many heme proteins 32 but mostly not over such large time spans (Supplementary Table 6 ). This large span of rebinding rates is likely to reflect a wide distribution of either enthalpic barriers reflecting different configurations of the dissociated heme and its environment (as assigned by Champion and coworkers in very recent work on the temperature dependence of heme-CO rebinding in CooA 33 and in free heme in solution 34 ) and/or of configurations of dissociated CO (in particular their orientations) in the heme pocket 35 . Heme binding to a flexible region of protein between TMD1 and NBD1 (Fig.…”

Adiabatic Ligand Binding in Heme Proteins: Ultrafast Kinetics of Methionine Rebinding in Ferrous Cytochrome c

“…On the other hand, the kinetic response below T g is clearly nonexponential and it can be attributed to a frozen ensemble of heme photoproduct conformations (as characterized by the heme out-of-plane equilibrium position). On the basis of the similarity between the Arrhenius prefactor found for this (Δ S = 2) reaction (∼10 11 s –1 ) and that of diatomic ligand binding (e.g., NO , and CO , ) to other heme proteins, with either Δ S = 1 or Δ S = 2 spin-state changes, we suggest that endogenous ligand binding in heme proteins is an adiabatic reaction with a spin-independent prefactor, as recently found for CO rebinding . Below 60 K, the measured rebinding kinetics are much faster than the prediction of the classical distributed coupling model, suggesting that either the heme photoproduct relaxation is somehow retarded or that quantum mechanical tunneling starts to compete with the classical “over-barrier reaction” at very low temperatures.…”

“…The time constant for Met80 rebinding in cyt c at room temperature (296 K) was found to be 7.2 ps, which is in good agreement with previous investigations. − Figure shows the kinetic traces of Met80 rebinding in cyt c below T g , where nonexponential behavior is clearly displayed. To fit these data, we invoked a model that involves a heme conformational distribution and which has been used successfully to explain CO rebinding kinetics in a variety of heme proteins and model compounds. ,,,,, …”

Modeling the dual oxygen- and pH-stimulated response of hemoglobin-loaded polyampholyte hydrogel for oxygen-pH coupled biosensor platform

“…To the best of our knowledge, no efforts were conducted experimentally and theoretically to investigate the performance of hemoglobin-loaded polyampholyte hydrogel as a function of ambient oxygen O2 coupled with environmental pH. In general, free hemoglobin consists of two components, the organic and inorganic parts [8][9][10], where the inorganic heme iron complex in the sensing domain of hemoglobin binds with oxygen O2 molecules [11][12][13], forming oxygen-heme iron complexes [14].…”