Evidence for a slow tertiary relaxation in the reaction of tert-butyl isocyanide with horseradish peroxidase.

The kinetics of tert-butyl isocyanide binding to the heme protein horseradish peroxidase (HRP) at 22 degrees C was examined on all time scales, from minutes to picoseconds, in aqueous borate buffer at pH 9.08. Unlike myoglobin (Mb) or hemoglobin, HRP shows two bimolecular ligand binding processes. For comparison, binding of the same ligand with Mb was measured under identical conditions. Ligand entry into the protein from the solvent in a mixing experiment is extremely slow in HRP: the bimolecular association constant is 0.04 M-1 s-1, while in Mb it is 4 x 10(3) M-1 s-1. Surprisingly, in view of that difference, picosecond and nanosecond photolyses reveal that once the ligand has reached the iron(II) site there is no difference in cage return or escape from the protein. The rate for the fastest cage return (from the contact pair) is close to 6 x 10(10) s-1 in both proteins. The rates of escape from the contact pair to form a secondary protein-caged pair are also similar: for Mb, 10 x 10(10) s-1, and for HRP, 8.5 x 10(10) s-1. The rate of rebinding from the protein-separated cage is near 4 x 10(6) s-1 in both proteins, and the rate of escape from protein to solvent is close to 3.7 x 10(6) s-1 in both. The difference between the two proteins lies in the low-millisecond time domain. After flash photolysis of HRP, there is a concentration-dependent recombination not seen in mixing experiments. This bimolecular rate constant varies slightly for different HRP preparations, being 2.6 x 10(4) or 4.0 x 10(4) M-1 s-1 in two cases, both of which are much faster than is observed in mixing experiments, namely, 0.04 M-1 s-1. In Mb, photolysis and mixing experiments consistently give the same combination rate, which is somewhat slower than the faster part of the HRP recombination. Similar measurements for the smaller ligand methyl isocyanide revealed no anomalous behavior. The interpretation proposed involves tertiary relaxation after ligand escape, which is significant in blocking the return of the large t-BuNC, but has no apparent effect on smaller ligands. Thus, HRP-t-BuNC reveals in dramatic fashion a phenomenon merely hinted at in earlier work involving the T-state binding kinetics of hemoglobin.

Myoglobin-NO at low pH: free four-coordinated heme in the protein pocket.

In either sperm whale or horse heart myoglobin, binding of NO and lowering of solution pH work together to weaken, and ultimately break, the bond between iron and the proximal histidine. This is reminiscent of the reaction observed at neutral pH in the case of guanylate cyclase, the heme enzyme that catalyzes the conversion of GTP to cGMP. Bond breaking is characterized by a spectral change from a nine-line to a three-line ESR signal and accompanied by a shift from 420 to 387 nm in the UV-vis spectrum of the Soret band maximum. Analysis of the pH-dependent spectral changes shows that they are reversible, at least within a few hours, that the transition is cooperative, involving six protons during pH lowering but only two as it is raised, and that the pK is about 4.7. Different proteins exhibit different pK values, which are generally lower than that for "chelated" protoheme. The pK differences reflect the extra bond stability afforded by the protein structure. Investigations of thermal and photochemical NO displacement by CO suggest that the local pocket around the ligand, although significantly altered (according to circular dichroism investigations), nonetheless still imposes a barrier against the outward diffusion of ligand into the solvent. Nanosecond and picosecond flash photolysis shows that in proteins at low pH there is an extremely efficient geminate recombination of the ligand with the four-coordinated species through a single-exponential process. This occurs to a significantly larger extent than for the case of NO-"chelated" protoheme (where no distal barrier for ligand is present). At neutral pH, when the proximal histidine bond is intact, the geminate recombination for NO takes longer and displays multiexponential kinetics. Altogether, these results suggest that, even though distal effects probably also play a role, proximal effects make an important contribution in modulating ligand-iron bond formation.

Geminate recombination of diatomic ligands CO, O2, and NO with myoglobin.

The kinetics of geminate recombination of horse heart myoglobin with the diatomic ligands carbon monoxide, dioxygen, and nitric oxide have been reexamined. The new measurements are distinguished from previous studies by (1) consideration of the complete time range longer than 1 ps, (2) inclusion of the effect of temperature changes near ambient, (3) attention to the relation between recombination kinetics and the yield of dissociated partners on the millisecond time scale, and (4) use of singular value decomposition in the analysis. These picosecond results, together with earlier nanosecond data, for O2 prove that models incorporating one, two, or even three discrete intermediates are not sufficient to account for all features of geminate recombination kinetics. Instead, a continuous evolution of the geminate pair distribution is preferred.

Quaternary structure and the geminate recombination of carp hemoglobin with methylisocyanide.

The kinetics of geminate recombination were studied for the methylisocyanide derivative of carp hemoglobin. Carp hemoglobin is of interest because it has been established that the fully liganded form switches between a high affinity R state at pH 9 and a low affinity T state at pH 6 in the presence of IHP. Geminate recombination was observed on both the picosecond and the nanosecond time scales under all conditions; however, only a small variation is observed in the rates and the yields of geminate recombination as the protein switches from the R to the T state. Taken together with overall "on" and "off" rates, the data indicate that the change from the R to the T configuration affects bond breaking most, but also influences subsequent escape from the protein as well as both entry into the protein and bond formation. There is some reason to postulate tertiary conformational change in the T state on the microsecond time scale following ligand escape from the protein.