Structural factors controlling ligand binding to myoglobin: A kinetic hole-burning study

Using temperature-derivative spectroscopy in the temperature range below 100 K, we have studied the dependence of the Soret band on the recombination barrier in sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation at 12 K. The spectra were separated into contributions from the photodissociated species, Mb*CO, and CO-bound myoglobin. The line shapes of the Soret bands of both photolyzed and liganded myoglobin were analyzed with a model that takes into account the homogeneous bandwidth, coupling of the electronic transition to vibrational modes, and static conformational heterogeneity. The analysis yields correlations between the activation enthalpy for rebinding and the model parameters that characterize the homogeneous subensembles within the conformationally heterogeneous ensemble. Such couplings between spectral and functional parameters arise when they both originate from a common structural coordinate. This effect is frequently denoted as “kinetic hole burning.” The study of these correlations gives direct insights into the structure–function relationship in proteins. On the basis of earlier work that assigned spectral parameters to geometric properties of the heme, the connections with the heme geometry are discussed. We show that two separate structural coordinates influence the Soret line shape, but only one of the two is coupled to the enthalpy barrier for rebinding. We give evidence that this coordinate, contrary to widespread belief, is not the iron displacement from the mean heme plane.

Solid and liquid phase 59Co NMR studies of cobalamins and their derivatives

We describe the application of 59Co NMR to the study of naturally occurring cobalamins. Targets of these investigations included vitamin B12, the B12 coenzyme, methylcobalamin, and dicyanocobyrinic acid heptamethylester. These measurements were carried out on solutions and powders of different origins, and repeated at a variety of magnetic field strengths. Particularly informative were the solid-state central transition NMR spectra, which when combined with numerical line shape analyses provided a clear description of the cobalt coupling parameters. These parameters showed a high sensitivity to the type of ligands attached to the metal and to the crystallization history of the sample. 59Co NMR determinations also were carried out on synthetic cobaloximes possessing alkyl, cyanide, aquo, and nitrogenated axial groups, substituents that paralleled the coordination of the natural compounds. These analogs displayed coupling anisotropies comparable to those of the cobalamins, as well as systematic up-field shifts that can be rationalized in terms of their stronger binding affinity to the cobalt atom. Cobaloximes also displayed a higher regularity in the relative orientations of their quadrupole and shielding coupling tensors, reflecting a higher symmetry in their in-plane coordination. For the cobalamines, poor correlations were observed between the values measured for the quadrupole couplings in the solid and the line widths observed in the corresponding solution 59Co NMR resonances.

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b595 was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the α-band of heme b595. The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b595 within a few ps, pointing to a direct interaction between hemes b595 and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b595 initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b595 provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b595 and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.