J
Am Chem Soc 123:576-587 (2001)
Nitrous oxide reductase (N
2OR) 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 (C
cO), 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
2OR 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
2OR 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'') RR spectrum exhibits
10
vibrational modes between 220 and 410 cm
-1,
with >1-cm
-1 S-34 isotope shifts
that sum
to -16.6 cm
-1. Many of these modes
are
also sensitive to Cu-65 and N-15(His) and, thus, can be assigned to
coupling
of the Cu-S stretch,
n (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 S-34 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 herefore suggest a
sulfur-bridged
cluster in Z
ox. The Z
ox
site
has its
n (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 (Zmv) with
six
S isotope-sensitive RR modes between 282 and 382 cm
-1
and a total S-34-shift of -16.9 cm
-1.
The
observation of a nearly identical RR spectrum in the C622D variant of N
2OR,
which lacks one of the conserved Cu-A Cys residues, establishes that
Cu-S
vibrations observed in this variant arise from the Zmv 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.