Ligand binding to synthetic mutant myoglobin (His-E7----Gly): role of the distal histidine.

D. Braunstein, A. Ansari, J. Berendzen, B. R. Cowen, K. D. Egeberg, H. Frauenfelder, M. K. Hong, P. Ormos, T. B. Sauke, R. Scholl

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Abstract

Low-temperature flash photolysis with IR and visible spectroscopy was used to probe the influence of the distal histidine His-64(E7) of sperm-whale myoglobin (Mb) on the orientation of bound carbon monoxide (CO) and on the kinetics of CO rebinding. The synthesis and high-level expression of a sperm-whale myoglobin gene in Escherichia coli permits the efficient substitution of the distal histidine through site-directed mutagenesis. Substitution of His-E7 with glycine [GlyE7]Mb bound with CO (CO[GlyE7]Mb) results in one broad bound-CO IR stretch band, v(C-O), centered at 1973 cm-1 at 10 K, in contrast to three distinct bands for native and synthetic wild-type MbCO at 1966, 1945, and 1929 cm-1. After flash photolysis at 10 K, the unbound state of CO[GlyE7]Mb exhibits two CO stretch bands, whereas MbCO has three. Fourier transform IR spectroscopy measurements of the linear dichroism after photoselective flash photolysis of CO bound to [GlyE7]Mb at 10 K reveals the bound CO to be oriented at an angle of alpha = 20 degrees +/- 2 degrees with respect to the heme normal. Flash photolysis data from 10 to 300 K provide evidence for a larger distal pocket and a smaller enthalpy barrier (by approximately 4 kJ/mol) for [GlyE7]MbCO as compared with wild-type MbCO. These results reinforce the notion that the dominant control of the binding step at the heme iron comes from the proximal side through the protein structure.

Original languageEnglish
Pages (from-to)8497-8501
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number22
DOIs
Publication statusPublished - Nov 1988

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    Braunstein, D., Ansari, A., Berendzen, J., Cowen, B. R., Egeberg, K. D., Frauenfelder, H., Hong, M. K., Ormos, P., Sauke, T. B., & Scholl, R. (1988). Ligand binding to synthetic mutant myoglobin (His-E7----Gly): role of the distal histidine. Proceedings of the National Academy of Sciences of the United States of America, 85(22), 8497-8501. https://doi.org/10.1073/pnas.85.22.8497