Contributions of Residue 45(CD3) and Heme-6-propionate to the Bimolecular and Geminate Recombination Reactions of Myoglobin

Theodore E. Carver, John S. Olson, Stephen J. Smerdon, Szymon Krzywda, Anthony J. Wilkinson, Quentin H. Gibson, Richard S. Blackmore, J. Dezz Ropp, Stephen G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

Overall association and dissociation rate constants were measured at 20 °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 (≤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 02 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 a 4-fold decrease in the geminate rate constant for the nanosecond rebinding of 02. The Arg45 to Glu substitution also caused a 4-fold decrease in the O2 affinity of pig myoglobin, which was due primarily to an increase in the overall oxygen dissociation rate constant. These results show that both the stability of the liganded, six-coordinate state and the geminate reactivity of unliganded, five-coordinate deoxymyoglobin are influenced by residue 45. The replacement of residue 45 with a polar or negatively charged amino acid caused substantial increases in the overall association rate constants for the binding of alkyl isocyanides. Elimination of the Arg45-propionate interactions by reconstitution of sperm whale myoglobin with protoheme IX dimethyl ester also enhanced the rate of alkyl isocyanide binding. These results suggest that the salt bridge between Arg45 or Lys45 and the heme-6-propionate does inhibit the movement of the larger isocyanide ligands into the distal pocket, presumably by restricting the motions of His64.

Original languageEnglish (US)
Pages (from-to)4697-4705
Number of pages9
JournalBiochemistry
Volume30
Issue number19
DOIs
StatePublished - May 1 1991

ASJC Scopus subject areas

  • Biochemistry

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