NATURE OF THE C-CLUSTER IN NI-CONTAINING CARBON-MONOXIDE DEHYDROGENASES

Citation
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
Citations number
46
Categorie Soggetti
Chemistry
ISSN journal
00027863
Volume
118
Issue
4
Year of publication
1996
Pages
830 - 845
Database
ISI
SICI code
0002-7863(1996)118:4<830:NOTCIN>2.0.ZU;2-R
Abstract
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.