Recent picosecond and nanosecond kinetic studies on the geminate recombination of photodissociated CO to Myoglobin, and Myoglobin mutants, has raised fundamental questions concerning the influence of different factors on the ligand binding processes to hemoproteins specifically, and to metalloproteins in general. We propose to use computational methods such as Density Functional theory, Molecular Mechanics, Molecular Dynamics, and Free Energy Perturbation calculations to study, and characterize, the factors which control ligand binding processes to Myoglobin (Mb) and the simple models thereof. We choose this group of systems because there exist a great deal of structural information on them; a requisite for using the proposed computational techniques. Specifically, we shall focus on the simulation of three factors as they affect the processes described above: 1) The Distal Steric effect, 2) The effect of local polarity, and 3) the effect of solvent. We will characterize these factors by using the AMBER software package to simulate both the equilibrium and kinetic properties of hemes, and Myoglobin. Briefly, the proposed methodology is summarized in the following steps: 1) Extend the current heme force field to the simulation of NO-heme and isocyanide-heme systems. 2) Reproduce trends in the binding affinities of ligands to hemes, Mb and Mb mutants via free-energy perturbation, and decompose these down into steric and electrostatic components. 3) Characterize the motion of the pertinent amino acids near the binding site via molecular dynamics. Initially these calculations will be run in vacuo, then the hemes in solvent, and finally the Myoglobin in solvent, which because of their size, will be done in a limited way. Based on preliminary work with simple heme-ligand systems, we anticipate that the major factors by which hemoproteins control the process of ligand binding are electrostatic, with Van der Waals interactions making a smaller contribution. How the relative importance of these two factors, and perhaps others, vary with binding- site structure, is an important question that needs to be addressed. Our studies will go a long way toward quantifying this structure-function relationship. Major applications of the answers to the questions we pose are in the fields of synthetic blood, activation of hemeproteins by NO, and sickle cell anemia. Training in the use of state-of-the-art molecular modeling software and graphics workstations is becoming essential for scientists today and certainly will be necessary for the next generation of scientists. Students will be trained in the planning, use, and interpretation of the calculations described herein and so will better prepare them for using these tools in the future.
