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Protein Science, Vol 3, Issue 11 2097-2103, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: Study of bacterial enzymes with cofactor substitutions and heme A model compounds

J. S. FELSCH, M. P. HORVATH, S. GURSKY, M. R. HOBAUGH, P. N. GOUDREAU, J. A. FEE, W. T. MORGAN, S. J. ADMIRAAL, M. IKEDA-SAITO, T. FUJIWARA, Y. FUKUMORI, T. YAMANAKA and R. A. COPELAND
Department of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, Illinois 60637

Second derivative absorption spectra are reported for the aa(3)-cytochrome c oxidase from bovine cardiac mitochondria, the aa(3)-600 ubiquinol oxidase from Bacillus subtilis, the ba(3)-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm. Taken together these data suggest that the observation of Soret splitting, including a feature at 450 nm, results from specific protein-cofactor interactions that are unique to the cytochrome a-binding pocket of the terminal oxidases. The conservation of this unique binding pocket among evolutionarily distant species may reflect some mechanistic significance for this structure.
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