Spectroscopic Computational and Kinetic Studies of the μ4-Sulfide Bridged Tetranuclear CuZ Cluster in N2O Reductase: pH Effect on the Edge Ligand and its Contribution to Reactivity

摘要

A combination of spectroscopy and DFT calculations has been used to evaluate the pH effect at the CuZ site in Pseudomonas nautica (Pn) N2OR and Achromobacter cycloclastes (Ac) N2OR and its relevance to catalysis. Absorption, MCD, EPR with sulfur K-edge XAS spectra of the enzymes at high and low pH show minor changes. However, resonance Raman (rR) spectroscopy of PnN2OR at high pH shows that the 415 cm⁻¹ Cu-S vibration (observed at low pH) shifts to higher frequency, loses intensity and obtains a 9 cm^{−1 18}O shift, implying significant Cu-O character, demonstrating the presence of a OH⁻ ligand at the CuICuIV edge. From DFT calculations both protonation of the OH⁻ to H2O or the μ4-S²⁻ to μ4-SH⁻ would produce large spectral changes which are not observed. Alternatively, DFT calculations including a lysine residue at an H-bonding distance from the CuICuIV edge ligand show that the position of the OH⁻ ligand depends on the protonation state of the lysine. This would change the coupling of the Cu-(OH) stretch with the Cu-S stretch, as observed in the rR spectrum. Thus the observed pH effect (pKa ~9.2) likely reflects protonation equilibrium of the lysine residue which would both raise E⁰ and provide a proton for lowering the barrier for the N-O cleavage and for reduction of the [Cu4S(im)7OH]²⁺ to the fully reduced 4Cu^I active form for turnover.