Nitrous oxide reductase (N2OR) from Pseudomonas stutzeri, a dimeric enzyme with a canonical metal ion content of at least six. Cu ions per subunit, contains two types of multinuclear copper sites: Cu-A and Cu-Z. An electron-transfer role for the dinuclear Cu-A Site is indicated based on its similarity to the Cu-A Site in cytochrome c oxidase (CcO), a dicysteinate-bridged, mixed-valence cluster. The Cu-Z site is the catalytic site, which had long been thought to have novel spectroscopic properties. However, the low-energy electronic transitions and resonance Raman features attributable to Cu-Z have been difficult to reconcile with a lack of conserved cysteine residues in standard;alignments of N2OR sequences, other than those associated with the Cu-A site. Recent evidence indicates that nitrous oxide reductase contains acid-labile sulfide and that this sulfide is a constituent of the Cu-Z site:(Rasmussen, T.; Berks, B. C.; Sanders-Loehr, J.; Dooley, D. M.; Zumft, W. G.; Thomson, A. J. Biochemistry 2000, 39, 12753-12756). We have used resonance Raman (RR) spectroscopy to selectively probe the Cu-A and Cu-Z sites of N2OR in three oxidation states (oxidized, semireduced, and reduced) as well as Cu-A only and Cu-Z only variants. The Cu-A (mixed-valence, also designated as A,,) RR spectrum exhibits 10 vibrational modes between 220 and 410 cm(-1), with >1-cm(-1) S-34 isotope shifts that sum to -16.6 cm(-1). Many of these modes are also sensitive to Cu-65 and N-15(His) and, thus, can be assigned to coupling of the Cu-S stretch, nu (Cu-S, with cysteine and histidine vibrations of the Cu(2)Cys(2)His(2) core.: The RR spectrum of the Cu-Z site (Z(ox)) reveals a novel Cu-sulfur chromophore with four S isotope-sensitive modes at 293, 347, 352, and 408 cm(-1), with a total S-34 shift of -19.9 cm(-1). The magnitude of the S isotope shifts and wide spread of perturbed frequencies are similar to those observed in Cu-A and therefore suggest a sulfur-bridged cluster in Z(ox). The Z(ox) site has its nu (Cu-S)-containing modes at higher energy and exhibits less mixing;: with ligand deformations, compared to Cu-A Reduction by dithionite produces a mixed-valence Cu-Z site (Z(mv)) with six S isotope-sensitive RR modes between 282 and 382 cm(-1) and a total S-34-shift of -16.9 cm(-1). The observation of a nearly identical RR spectrum in the C622D variant of N2OR, which lacks one of the conserved Cu-A Cys residues, establishes that Cu-S vibrations observed in this variant arise from the Z(mv) site. Furthermore, none of the features assigned to Cu-Z are detected in a second variant that contains only Cu-A Therefore the resonance Raman spectra reported here provide compelling evidence for a unique Cu-S cluster in the catalytic site of nitrous oxide reductase.