Contributions of residue 45(CD3) and heme-6-propionate to the bimolecular and geminate recombination reactions of myoglobin

Abstract

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)

Application of molecular dynamics and free energy perturbation methods to metalloporphyrin‐ligand systems ii: Co and dioxygen binding to myoglobin

“…It has been suggested that this residue acts as a gate for incoming ligands (Lambright et al, 1989;Carver et al, 1991). Aside from the His F8/por residue, the Arg CD3 residue has the largest energy contribution due to the mutation of the distal histidine (2 + 4, Table 4; 1 -+ 3, Table 5).…”

CO Rebinding to Protoheme: Investigations of the Proximal and Distal Contributions to the Geminate Rebinding Barrier

“…Thus, the difference in the entropic contribution to the distal free energy barrier in eq 5 is given by (7) where S D (Mb) = k ln Ω Mb and S D (+Im) = k ln Ω +Im with Ω denoting the number of possible distal pocket states associated with the CO ligand in its initial configuration prior to geminate recombination. Thus, eq 7 leads directly to (8) We now take the number of accessible initial states for the CO ligand in Mb, Ω Mb , to be proportional to the volume of the region from which the CO ligand must return during the geminate rebinding process. Similarly, the number of possible reactant states for geminate rebinding of the CO ligand to 2-MeImFePPIX in glycerol solvent, Ω +Im , is proportional to the volume of the glycerol solvent that is displaced by the photolyzed CO ligand.…”

“…[1][2][3][4][5] The diatomic ligand binds to the heme iron from the distal side, while an amino acid residue from the protein matrix (often histidine or cysteine) binds from the other (proximal) side. The dynamics associated with ligand binding to the heme group has been studied for many years in both native and mutant proteins [3][4][5][6][7][8][9][10][11][12][13][14][15] and in heme model compounds. [16][17][18][19][20][21][22][23][24] The binding process for model compounds involves at least two steps: the diffusion of ligand through the surrounding solvent or buffer followed by the bond formation step between the diatomic ligand and the iron.…”

Perturbations of the distal heme pocket in human myoglobin mutants probed by infrared spectroscopy of bound CO: correlation with ligand binding kinetics.

“…Extensive measurements of the ligand rebinding kinetics in a wide range of human and sperm whale (SW) Mb mutants demonstrate enormous variations in both the geminate and bimolecular binding kinetics (15)(16)(17)(18)(19). Although trends can be discerned, it is often difficult to understand the effects in terms of specific interactions.…”