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Spectroscopic and computational methods have been used to determine the protonation state of the edge sulfur ligand in the Cu 4 S 2 Cu Z form of the active site of nitrous oxide reductase (N 2 OR) in its 3Cu I Cu II (1-hole) and 2Cu I 2Cu II (2-hole) redox states. The EPR, absorption, and MCD spectra of 1-hole Cu Z indicate that the unpaired spin in this site is evenly delocalized over Cu I , Cu II , and Cu IV . 1-hole Cu Z is shown to have a μ 2 -thiolate edge ligand from the observation of S-H bending modes in the resonance Raman spectrum at 450 and 492 cm -1 that have significant deuterium isotope shifts (-137 cm -1 ) and are not perturbed up to pH 10. 2-hole Cu Z is characterized with absorption and resonance Raman spectroscopies as having two Cu-S stretching vibrations that profile differently. DFT models of the 1-hole and 2-hole Cu Z sites are correlated to these spectroscopic features to determine that 2-hole Cu Z has a μ 2 -sulfide edge ligand at neutral pH. The slow two electron (+1 proton) reduction of N 2 O by 1-hole Cu Z is discussed and the possibility of a reaction between 2-hole Cu Z and O 2 is considered.

Protonation state of the Cu4S2 CuZ site in nitrous oxide reductase: redox dependence and insight into reactivity

Protonation state of the Cu₄S₂ Cu_Z site in nitrous oxide reductase: redox dependence and insight into reactivity