Zg. Hu et al., NATURE OF THE C-CLUSTER IN NI-CONTAINING CARBON-MONOXIDE DEHYDROGENASES, Journal of the American Chemical Society, 118(4), 1996, pp. 830-845
The C-cluster of carbon monoxide dehydrogenase (CODH) appears to be th
e active site for the oxidation of CO to CO2. We have studied with EPR
and Mossbauer spectroscopy the enzymes from Rhodospirillum rubrum (CO
DHRr; similar to 8 Fe atoms and 1 Ni atom per alpha) and Clostridium t
hemzoaceticum (CODHCt; similar to 12 Fe atoms and 2 Ni atoms per alpha
beta). The study of CODHRr offers two advantages. First, the enzyme l
acks the A-cluster responsible for the synthase activity of CODHCt. Se
cond, a Ni-deficient protein (Ni-deficient CODHRr) containing all Fe c
omponents of the holoenzyme can be isolated. The holoenzymes of both s
pecies can be prepared in a state for which the C-cluster exhibits the
so-called g(av) = 1.82 EPR signal (C-red1); the spectra of Ni-deficie
nt CODHRr do not exhibit this signal. Our results are as follows: The
Mossbauer data show that all iron atoms of Ni-deficient CODHRr belong
to two [Fe4S4](1+/2+) clusters. The so-called B-cluster, which functio
ns in electron transfer, is diamagnetic in the [Fe4S4](2+) state, B-ox
, and exhibits an S = 1/2 (g = 1.94) EPR signal in the [Fe4S4](+) stat
e, B-red. The spectroscopic properties of the B-cluster are the same i
n Ni-deficient, holo-CODHRr and CODHCt. The precursor to the C-cluster
of Ni-deficient CODHRr, labeled C, is diamagnetic in the [Fe4S4](2+)
state, but has an S = 3/2 spin in the [Fe4S4](+) form. Upon incorpora
tion of Ni, the properties of the C-cluster change substantially. At
E(m)(,) = -110 mV, the C-cluster undergoes a 1-electron reduction from
the oxidized state, C-ox, to the reduced state, C-red1, which exhibit
s the g(av) = 1.82 EPR signal. A study of a sample poised at -300 mV s
hows that this signal originates from an S = 1/2 [Fe4S4](+) cluster. I
n this state, the cluster has a distinct subsite, ferrous component II
(FCII), having Delta E(Q) = 2.82 mm/s and delta = 0.82 mm/s; these pa
rameters suggest a pentacoordinate site somewhat similar to subsite Fe
-a of the Fe4S4 cluster of active aconitase. The same values for Delta
E(Q) and delta were observed for CODHCt. Upon addition of CN-, a pote
nt inhibitor of CO oxidation, the Delta E(Q) of FCII of CODHCt changes
from 2.82 to 2.53 mm/s, suggesting that CN- binds to the FCII iron. T
he Mossbauer studies of CODHRr showed that only similar to 60% of the
C-clusters were capable of attaining the C-red1 state; the remainder w
ere C-ox (or C-ox()). For the Mossbauer sample, the EPR spin concentr
ation of the g(av) = 1.82 signal was similar to 65% of that determined
for the g = 1.94 signal of B(red)of the fully reduced sample, a resul
t consistent with the similar to 60% obtained from Mossbauer spectrosc
opy. When CODHRr was reduced with CO or dithionite, a fraction of the
C-clusters developed a signal similar to the g(av) = 1.86 signal (C-re
d2) of CODHCt. The Mossbauer and EPR spectra of dithionite-reduced COD
HRr show that a large fraction of the C-centers are in a state for whi
ch the [Fe4S4](+) cluster has S = 3/2. While the assumption of an [Fe4
S4](+) cluster with an aconitase-type subsite electronically isolated
from the Ni site can explain the g values of the g(av) = 1.82 signal a
nd the absence of Ni-61 hyperfine interactions, published resonance Ra
man and EPR data suggest that the Ni site may be electronically linked
to the Fe-S moiety of the C-cluster. We present a model that consider
s a weak exchange interaction (effective coupling constant j) between
an S = 1 Ni-II site (zero-field splitting, D) and the S = 1/2 ground s
tate of the [Fe4S4](+) cluster. This model suggests \j\ < 2 cm(-1), ac
counts for the g values of C-red1, and provides an explanation for the
unusual g values (g(av) approximate to 2.16) reported by S. W. Ragsda
le and co-workers for the adducts of CODHCt with thiocyanate and cyana
te. The coupling model is consistent with Ni-61 EPR studies of CODH.