THE PURPLE MIXED-VALENCE CUA CENTER IN NITROUS-OXIDE REDUCTASE - EPR OF THE COPPER-63-ENRICHED, COPPER-65-ENRICHED, AND BOTH COPPER-65-ENRICHED AND [N-15]HISTIDINE-ENRICHED ENZYME AND A MOLECULAR-ORBITAL INTERPRETATION

EPR spectra for the purple mixed-valence [Cu-1.5+...Cu-1.5+], S = 1/2, Site (Cu-A) in nitrous-oxide reductase (N(2)OR) were obtained after i nsertion of either Cu-63 or Cu-65 Or both Cu-65 and [N-15]histidine. T he spectrum of Cu-65- and [N-15]histidine-enriched N(2)OR improved the resolution of the Cu hyperfine lines, but no lines from nitrogen and proton couplings were resolved. The Cu hyperfine parameters obtained b y a theory analogous to that of Maki and McGarvey were indicative of a highly covalent Cu site. The total Cu character (Cu-A1 + Cu-A2) in th e ground state wave function required to describe the spin density dis tribution was 31-37% compared to 41% for type-1 Cu in plastocyanin. Th is value does not completely account for the reduction of g(max) from 2.23 of type-1 Cu in plastocyanin to 2.18 of Cu-A. Remaining discrepan cies were discussed in terms of different alignments of the principal axes for the hypothetical monomeric Cu-A1 and Cu-A2 in [Cu-1.5+...Cu-1 .5+]. This effect appeared in the simulations of the EPR spectra as a noncoincidence between the Cu hyperfine and g principal axis systems. The g-value analysis of Cu-A predicts an electric dipole forbidden abs orption band in the near-infrared region. Based on X-ray structural da ta of Cu-A in cytochrome c oxidase, iterative extended Huckel and UHF- INDO/S calculations on a sulfur-bridged [(NH3)Cu-1.5+(SCH3)(2)Cu-1.5+( NH3)](+) core were used to interpret the EPR results. The ground state was selected as B-2(3u) and not B-2(2u), because B-2(2u) has very lit tle spin density (<0.5%) on the coordinated nitrogen atoms, which cont radicts the experimental value of 3-5% spin density found on N-His.