title: Spectroscopic, computational, and kinetic studies of the mu4-sulfide-bridged tetranuclear CuZ cluster in N2O reductase: pH effect on the edge ligand and its contribution to reactivity.
abstract: A combination of spectroscopy and density functional theory (DFT) calculations has been used to evaluate the pH effect at the CuZ site in Pseudomonas nautica (Pn) nitrous oxide reductase (N2OR) and Achromobacter cycloclastes (Ac) N2OR and its relevance to catalysis. Absorption, magnetic circular dichroism, and electron paramagnetic resonance with sulfur K-edge X-ray absorption 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 18O shift, implying significant Cu-O character, demonstrating the presence of a OH- ligand at the CuICuIV edge. From DFT calculations, protonation of either the OH- to H2O or the mu4-S2- to mu4-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 approximately 9.2) likely reflects protonation equilibrium of the lysine residue, which would both raise E degrees 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 4CuI active form for turnover.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Somdatta
authors.initials: S
authors.lastname: Ghosh
authors.forename: Serge I
authors.initials: SI
authors.lastname: Gorelsky
authors.forename: Serena DeBeer
authors.initials: SD
authors.lastname: George
authors.forename: Jeannine M
authors.initials: JM
authors.lastname: Chan
authors.forename: Inês
authors.initials: I
authors.lastname: Cabrito
authors.forename: David M
authors.initials: DM
authors.lastname: Dooley
authors.forename: José J G
authors.initials: JJ
authors.lastname: Moura
authors.forename: Isabel
authors.initials: I
authors.lastname: Moura
authors.forename: Edward I
authors.initials: EI
authors.lastname: Solomon
mesh_terms: D003198:Computer Simulation; D003300:Copper / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D013440:Sulfides / Q000737:chemistry*; D014732:Vibration
chemical_list: D003432:Cross-Linking Reagents; D008024:Ligands; D013440:Sulfides; D003300:Copper; D010088:Oxidoreductases; C022763:nitrous oxide reductase