The gateway to the active site of heme-copper oxidases

The spectroscopy and dynamics of CO binding were measured for wild-type and mutant cytochromes bo, members of the superfamily of heme-copper oxidases. The results suggest that access of ligands, including substrate O2, to the binuclear Fe-Cu active site is controlled at two levels. CO recombination...

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Bibliographic Details
Published in:Biochemistry (Easton) 1993-11, Vol.32 (45), p.11953-11956
Main Authors: Lemon, Douglas D, Calhoun, Melissa W, Gennis, Robert B, Woodruff, William H
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Binding Sites
Biological and medical sciences
Copper
Cytochrome b Group
Cytochromes - metabolism
Enzymes and enzyme inhibitors
Escherichia coli
Escherichia coli Proteins
Fundamental and applied biological sciences. Psychology
Hemeproteins - metabolism
Kinetics
Oxidoreductases
Spectroscopy, Fourier Transform Infrared
Substrate Specificity
Temperature
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Summary:The spectroscopy and dynamics of CO binding were measured for wild-type and mutant cytochromes bo, members of the superfamily of heme-copper oxidases. The results suggest that access of ligands, including substrate O2, to the binuclear Fe-Cu active site is controlled at two levels. CO recombination to the wild-type ubiquinol oxidase exhibited saturation kinetics (kmax = 190 s-1, Km = 2.4 mM), indicative of the existence of an intermediate in the ligand-binding pathway. FTIR spectroscopy and TRIR spectroscopy were used to demonstrate conclusively that this intermediate was a CuB-CO complex. Two mutant oxidases (His333Leu, His334Leu) which lack CuB showed no evidence of saturation of CO rebinding, even up to 21 mM CO. Also, the absolute rates of CO binding to the mutant oxidases were much greater than for wild type, even at CO concentrations well below the apparent Km for wild-type enzyme. These results clearly indicate that the copper ion at the binuclear site acts as an obligatory way station, or gate, severely limiting the approach of ligands to the heme active site. Further, an analysis of the rate constants for CO binding to CuB suggests that the protein structure external to the binuclear site regulates ligand entry into this site. We propose that these control mechanisms for substrate binding are operative throughout this general class of enzymes.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00096a002