Spectroscopic, Computational, and Kinetic Studies of the Mu-Sulfide-Bridged Tetranuclear CuZ Cluster in N(2)O 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 Cu{sub Z} site in Pseudomonas nautica (Pn) nitrous oxide reductase (N{sub 2}OR) and Achromobacter cycloclastes (Ac) N{sub 2}OR 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 PnN{sub 2}OR at high pH shows that the 415 cm{sup -1} Cu-S vibration (observed at low pH) shifts to higher frequency, loses intensity, and obtains a 9 cm{sup -1} {sup 18}O shift, implying significant Cu-O character, demonstrating the presence of a OH{sup -} ligand at the Cu{sub I}Cu{sub IV} edge. From DFT calculations, protonation of either the OH{sup -} to H{sub 2}O or the {mu}{sub 4}-S{sup 2-} to {mu}{sub 4}-SH{sup -} would produce large spectral changes which are not observed. Alternatively, DFT calculations including a lysine residue at an H-bonding distance from the Cu{sub I}Cu{sub IV} edge ligand show that the position of the OH{sup -} 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 (pK{sub a} {approx} 9.2) likely reflects protonation equilibrium of the lysine residue, which would both raise E{sup o} and provide a proton for lowering the barrier for the N-O cleavage and for reduction of the [Cu{sub 4}S(im){sub 7}OH]{sup 2+} to the fully reduced 4Cu{sup I} active form for turnover.
- Research Organization:
- SLAC National Accelerator Lab., Menlo Park, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC02-76SF00515
- OSTI ID:
- 1030545
- Report Number(s):
- SLAC-REPRINT-2011-101; JACSAT; TRN: US201124%%411
- Journal Information:
- Journal of the American Chemical Society, Vol. 129, Issue 13; ISSN 0002-7863
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
60 APPLIED LIFE SCIENCES
ABSORPTION
ABSORPTION SPECTRA
CATALYSIS
CLEAVAGE
DENSITY FUNCTIONAL METHOD
ELECTRON SPIN RESONANCE
ENZYMES
KINETICS
LYSINE
MAGNETIC CIRCULAR DICHROISM
NITROUS OXIDE
OXIDOREDUCTASES
PH VALUE
PSEUDOMONAS
RESIDUES
RESONANCE
SPECTROSCOPY
SULFUR
Other
OTHER