PMID- 11456570
OWN - NLM
STAT- MEDLINE
DCOM- 20010920
LR  - 20190708
IS  - 0002-7863 (Print)
IS  - 0002-7863 (Linking)
VI  - 123
IP  - 4
DP  - 2001 Jan 31
TI  - Characterization of the copper-sulfur chromophores in nitrous oxide reductase by 
      resonance raman spectroscopy: evidence for sulfur coordination in the catalytic 
      cluster.
PG  - 576-87
AB  - Nitrous oxide reductase (N(2)OR) from Pseudomonas stutzeri, a dimeric enzyme with 
      a canonical metal ion content of at least six Cu ions per subunit, contains two 
      types of multinuclear copper sites: Cu(A) and Cu(Z). An electron-transfer role 
      for the dinuclear Cu(A) site is indicated based on its similarity to the Cu(A) 
      site in cytochrome c oxidase (CcO), a dicysteinate-bridged, mixed-valence 
      cluster. The Cu(Z) site is the catalytic site, which had long been thought to 
      have novel spectroscopic properties. However, the low-energy electronic 
      transitions and resonance Raman features attributable to Cu(Z) have been 
      difficult to reconcile with a lack of conserved cysteine residues in standard 
      alignments of N(2)OR sequences, other than those associated with the Cu(A) site. 
      Recent evidence indicates that nitrous oxide reductase contains acid-labile 
      sulfide and that this sulfide is a constituent of the Cu(Z) site (Rasmussen, T.; 
      Berks, B. C.; Sanders-Loehr, J.; Dooley, D. M.; Zumft, W. G.; Thomson, A. J. 
      Biochemistry 2000, 39, 12753-12756). We have used resonance Raman (RR) 
      spectroscopy to selectively probe the Cu(A) and Cu(Z) sites of N(2)OR in three 
      oxidation states (oxidized, semireduced, and reduced) as well as Cu(A)-only and 
      Cu(Z)-only variants. The Cu(A) (mixed-valence, also designated as A(mv)) RR 
      spectrum exhibits 10 vibrational modes between 220 and 410 cm(-1), with >1-cm(-1) 
      (34)S isotope shifts that sum to -16.6 cm(-1). Many of these modes are also 
      sensitive to (65)Cu and (15)N(His) and, thus, can be assigned to coupling of the 
      Cu-S stretch, nu(Cu-S), with cysteine and histidine vibrations of the 
      Cu(2)Cys(2)His(2) core. The RR spectrum of the Cu(Z) site (Z(ox)) reveals a novel 
      Cu-sulfur chromophore with four S isotope-sensitive modes at 293, 347, 352, and 
      408 cm(-1), with a total (34)S shift of -19.9 cm(-)(1). The magnitude of the S 
      isotope shifts and wide spread of perturbed frequencies are similar to those 
      observed in Cu(A) and therefore suggest a sulfur-bridged cluster in Z(ox). The 
      Z(ox) site has its nu(Cu-S)-containing modes at higher energy and exhibits less 
      mixing with ligand deformations, compared to Cu(A). Reduction by dithionite 
      produces a mixed-valence Cu(Z) site (Z(mv)) with six S isotope-sensitive RR modes 
      between 282 and 382 cm(-1) and a total (34)S-shift of -16.9 cm(-1). The 
      observation of a nearly identical RR spectrum in the C622D variant of N(2)OR, 
      which lacks one of the conserved Cu(A) Cys residues, establishes that Cu-S 
      vibrations observed in this variant arise from the Z(mv) site. Furthermore, none 
      of the features assigned to Cu(Z) are detected in a second variant that contains 
      only Cu(A). Therefore the resonance Raman spectra reported here provide 
      compelling evidence for a unique Cu-S cluster in the catalytic site of nitrous 
      oxide reductase.
FAU - Alvarez, M L
AU  - Alvarez ML
AD  - Department of Chemistry and Biochemistry, Montana State University, Bozeman, 
      Montana 59717, USA.
FAU - Ai, J
AU  - Ai J
FAU - Zumft, W
AU  - Zumft W
FAU - Sanders-Loehr, J
AU  - Sanders-Loehr J
FAU - Dooley, D M
AU  - Dooley DM
LA  - eng
GR  - GM-18865/GM/NIGMS NIH HHS/United States
GR  - RR0223/RR/NCRR NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Am Chem Soc
JT  - Journal of the American Chemical Society
JID - 7503056
RN  - 0 (Bacterial Proteins)
RN  - 0 (Ligands)
RN  - 4QD397987E (Histidine)
RN  - 70FD1KFU70 (Sulfur)
RN  - 789U1901C5 (Copper)
RN  - EC 1.- (Oxidoreductases)
RN  - EC 1.7.2.4 (nitrous oxide reductase)
SB  - IM
MH  - Bacterial Proteins/chemistry
MH  - Catalytic Domain
MH  - Copper/chemistry
MH  - Histidine/chemistry
MH  - Ligands
MH  - Models, Molecular
MH  - Oxidation-Reduction
MH  - Oxidoreductases/*chemistry
MH  - Pseudomonas/enzymology
MH  - Soil Microbiology
MH  - Spectrum Analysis, Raman
MH  - Sulfur/chemistry
EDAT- 2001/07/18 10:00
MHDA- 2001/09/21 10:01
CRDT- 2001/07/18 10:00
PHST- 2001/07/18 10:00 [pubmed]
PHST- 2001/09/21 10:01 [medline]
PHST- 2001/07/18 10:00 [entrez]
AID - ja994322i [pii]
AID - 10.1021/ja994322i [doi]
PST - ppublish
SO  - J Am Chem Soc. 2001 Jan 31;123(4):576-87. doi: 10.1021/ja994322i.