Nature of the C-Cluster in Ni-Containing Carbon Monoxide Dehydrogenases
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- 作者：Zhengguo Hu ; Nathan J. Spangler ; Mark E. Anderson ; Jinqiang Xia ; Paul W. Ludden ; Paul A. Lindahl ; and Eckard Mü ; nck
- 刊名：Journal of the American Chemical Society
- 出版年：1996
- 出版时间：January 31, 1996
- 年：1996
- 卷：118
- 期：4
- 页码：830 - 845
- 全文大小：324K
- 年卷期：v.118,no.4(January 31, 1996)
- ISSN：1520-5126
文摘
The C-cluster of carbon monoxide dehydrogenase (CODH)appears to be the active site for the oxidationof CO to CO2. We have studied with EPR andMössbauer spectroscopy the enzymes from Rhodospirillumrubrum(CODHRr; ~8 Fe atoms and 1 Ni atom per 
) andClostridium thermoaceticum (CODHCt; ~12 Featoms and 2 Niatoms per 
). The study of CODHRr offers twoadvantages. First, the enzyme lacks the A-cluster responsibleforthe synthase activity of CODHCt. Second, aNi-deficient protein (Ni-deficient CODHRr) containing allFe componentsof the holoenzyme can be isolated. The holoenzymes of both speciescan be prepared in a state for which theC-cluster exhibits the so-called gav = 1.82EPR signal (Cred1); the spectra of Ni-deficientCODHRr do not exhibit thissignal. Our results are as follows: The Mössbauer datashow that all iron atoms of Ni-deficient CODHRr belongtotwo [Fe4S4]1+/2+ clusters.The so-called B-cluster, which functions in electron transfer, isdiamagnetic in the[Fe4S4]2+state, Box, and exhibits an S = 1/2(g = 1.94) EPR signal in the[Fe4S4]+ state,Bred. The spectroscopic propertiesof the B-cluster are the same in Ni-deficient, holo-CODHRrand CODHCt. The precursor to the C-cluster ofNi-deficient CODHRr, labeled C*, is diamagnetic in the[Fe4S4]2+ state, but has anS = 3/2 spin in the[Fe4S4]+ form.Upon incorporation of Ni, the properties of the C*-cluster changesubstantially. At E'm = -110 mV, theC-clusterundergoes a 1-electron reduction from the oxidized state,Cox, to the reduced state, Cred1, whichexhibits the gav =1.82 EPR signal. A study of a sample poised at -300 mV showsthat this signal originates from an S = 1/2[Fe4S4]+cluster. In this state, the cluster has a distinct subsite,ferrous component II (FCII), having 
EQ =2.82 mm/s and
= 0.82 mm/s; these parameters suggest a pentacoordinate sitesomewhat similar to subsite Fea of theFe4S4 clusterof active aconitase. The same values forEQ and were observed forCODHCt. Upon addition of CN-, apotentinhibitor of CO oxidation, the EQ of FCII ofCODHCt changes from 2.82 to 2.53 mm/s, suggesting thatCN- bindsto the FCII iron. The Mössbauer studies ofCODHRr showed that only ~60% of the C-clusters werecapable ofattaining the Cred1 state; the remainder wereCox (or C*ox). For the Mössbauersample, the EPR spin concentrationof the gav = 1.82 signal was ~65% of thatdetermined for the g = 1.94 signal of Bred ofthe fully reduced sample,a result consistent with the ~60% obtained from Mössbauerspectroscopy. When CODHRr was reduced with COordithionite, a fraction of the C-clusters developed a signal similar tothe gav = 1.86 signal (Cred2) ofCODHCt. TheMössbauer and EPR spectra of dithionite-reducedCODHRr show that a large fraction of the C-centers are in astatefor which the [Fe4S4]+ clusterhas S = 3/2. While the assumption of an[Fe4S4]+ cluster with anaconitase-typesubsite electronically isolated from the Ni site can explain theg values of the gav = 1.82 signaland the absence of61Ni hyperfine interactions, published resonance Ramanand EPR data suggest that the Ni site may beelectronicallylinked to the Fe-S moiety of the C-cluster. We present a modelthat considers a weak exchange interaction (effectivecoupling constant j) between an S = 1NiII site (zero-field splitting, D) and theS = 1/2 ground state of the[Fe4S4]+cluster. This model suggests 
j < 2cm-1, accounts for the g values ofCred1, and provides an explanation for theunusual g values (gav 
2.16)reported by S. W. Ragsdale and co-workers for the adducts ofCODHCt with thiocyanateand cyanate. The coupling model is consistent with61Ni EPR studies of CODH.
) andClostridium thermoaceticum (CODHCt; ~12 Featoms and 2 Niatoms per ). The study of CODHRr offers twoadvantages. First, the enzyme lacks the A-cluster responsibleforthe synthase activity of CODHCt. Second, aNi-deficient protein (Ni-deficient CODHRr) containing allFe componentsof the holoenzyme can be isolated. The holoenzymes of both speciescan be prepared in a state for which theC-cluster exhibits the so-called gav = 1.82EPR signal (Cred1); the spectra of Ni-deficientCODHRr do not exhibit thissignal. Our results are as follows: The Mössbauer datashow that all iron atoms of Ni-deficient CODHRr belongtotwo [Fe4S4]1+/2+ clusters.The so-called B-cluster, which functions in electron transfer, isdiamagnetic in the[Fe4S4]2+state, Box, and exhibits an S = 1/2(g = 1.94) EPR signal in the[Fe4S4]+ state,Bred. The spectroscopic propertiesof the B-cluster are the same in Ni-deficient, holo-CODHRrand CODHCt. The precursor to the C-cluster ofNi-deficient CODHRr, labeled C*, is diamagnetic in the[Fe4S4]2+ state, but has anS = 3/2 spin in the[Fe4S4]+ form.Upon incorporation of Ni, the properties of the C*-cluster changesubstantially. At E'm = -110 mV, theC-clusterundergoes a 1-electron reduction from the oxidized state,Cox, to the reduced state, Cred1, whichexhibits the gav =1.82 EPR signal. A study of a sample poised at -300 mV showsthat this signal originates from an S = 1/2[Fe4S4]+cluster. In this state, the cluster has a distinct subsite,ferrous component II (FCII), having EQ =2.82 mm/s and = 0.82 mm/s; these parameters suggest a pentacoordinate sitesomewhat similar to subsite Fea of theFe4S4 clusterof active aconitase. The same values forEQ and were observed forCODHCt. Upon addition of CN-, apotentinhibitor of CO oxidation, the EQ of FCII ofCODHCt changes from 2.82 to 2.53 mm/s, suggesting thatCN- bindsto the FCII iron. The Mössbauer studies ofCODHRr showed that only ~60% of the C-clusters werecapable ofattaining the Cred1 state; the remainder wereCox (or C*ox). For the Mössbauersample, the EPR spin concentrationof the gav = 1.82 signal was ~65% of thatdetermined for the g = 1.94 signal of Bred ofthe fully reduced sample,a result consistent with the ~60% obtained from Mössbauerspectroscopy. When CODHRr was reduced with COordithionite, a fraction of the C-clusters developed a signal similar tothe gav = 1.86 signal (Cred2) ofCODHCt. TheMössbauer and EPR spectra of dithionite-reducedCODHRr show that a large fraction of the C-centers are in astatefor which the [Fe4S4]+ clusterhas S = 3/2. While the assumption of an[Fe4S4]+ cluster with anaconitase-typesubsite electronically isolated from the Ni site can explain theg values of the gav = 1.82 signaland the absence of61Ni hyperfine interactions, published resonance Ramanand EPR data suggest that the Ni site may beelectronicallylinked to the Fe-S moiety of the C-cluster. We present a modelthat considers a weak exchange interaction (effectivecoupling constant j) between an S = 1NiII site (zero-field splitting, D) and theS = 1/2 ground state of the[Fe4S4]+cluster. This model suggests j < 2cm-1, accounts for the g values ofCred1, and provides an explanation for theunusual g values (gav 2.16)reported by S. W. Ragsdale and co-workers for the adducts ofCODHCt with thiocyanateand cyanate. The coupling model is consistent with61Ni EPR studies of CODH.