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.
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.
Ghosh
Somdatta
S
Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Gorelsky
Serge I
SI
George
Serena DeBeer
SD
Chan
Jeannine M
JM
Cabrito
Inês
I
Dooley
David M
DM
Moura
José J G
JJ
Moura
Isabel
I
Solomon
Edward I
EI
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
D003432:Cross-Linking Reagents; D008024:Ligands; D013440:Sulfides; D003300:Copper; D010088:Oxidoreductases; C022763:nitrous oxide reductase