Temperature-dependent heme kinetics with nonexponential binding and barrier relaxation in the absence of protein conformational substates

Abstract

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

Coherence Spectroscopy Investigations of the Low-Frequency Vibrations of Heme: Effects of Protein-Specific Perturbations

“…28,63,64 For example, heme ruffling has been suggested 65 to be a distortion that is involved in stabilizing the net positive charge of the ferric heme in nitrophorin (NP4). The general picture that needs to be explored is whether or not the protein matrix interacts with the normally planar heme and causes it to adopt conformations that enhance or retard specific reactions.…”

“…67 Recent kinetic studies have revealed a significant amount of inhomogeneity that is inherent to the heme group in solution, but it appears that the inhomogeneous distributions are some-what larger in the protein. 28 Further experimental testing of these ideas will require temperature-dependent measurements of the coherence spectra of heme model compounds.…”

“…28,[69][70][71][72][73][74][75] To further investigate the interactions between the heme chromophore and its surroundings, we carried out a series of temperature-dependent FCS measurements on the ferrous HRP species. We have reported similar FCS studies on deoxyMb 40 and the current data is consistent with the earlier work.…”

“…34 The effect of the distortions is to lower the inherent symmetry of the heme and activate out-of-plane low-frequency modes that fall in the region ≲k B T. These modes are likely to play a role in heme protein function, either by helping to "trap" product states once they are formed 35 or as direct reaction coordinates. 28 …”

“…The theoretical details of this time domain spectroscopic technique can be found elsewhere. [19][20][21][22][23][24][25][26][27] One important advantage of the FCS technique over more traditional frequency domain vibrational spectroscopies is its ability to monitor very low-frequency vibrational modes (20- 28 To assess the importance of the protein environment on the low-frequency vibrational spectrum of the heme, we carried out a series of experiments on Mb, HRP (isoenzyme C), and Cgb. Each of these heme proteins has a b-type heme in the active site that is covalently bound to the rest of the protein through a histidine amino acid (His 93 in Mb, His170 in HRP, and His85 in Cgb).…”