The Purple Mixed-Valence Cu_A Center in Nitrous-oxide Reductase:  EPR of the Copper-63-, Copper-65-, and Both Copper-65- and [¹⁵N]Histidine-Enriched Enzyme and a Molecular Orbital Interpretation

Abstract

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 insertion of either 63 Cu or 65 Cu or both 65 Cu and [ 15 N]histidine. The spectrum of 65 Cu- and [ 15 N]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 by 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 the ground state wave function required to describe the spin density distribution was 31−37% compared to 41% for type-1 Cu in plastocyanin. This 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 discrepancies 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 absorption band in the near-infrared region. Based on X-ray structural data of Cu A in cytochrome c oxidase, iterative extended Hückel and UHF-INDO/S calculations on a sulfur-bridged [(NH 3 )Cu 1.5+ (SCH 3 ) 2 Cu 1.5+ (NH 3 )] + core were used to interpret the EPR results. The ground state was selected as 2 B 3u and not 2 B 2u, because 2 B 2u has very little spin density (<0.5%) on the coordinated nitrogen atoms, which contradicts the experimental value of 3−5% spin density found on N His .