Nature of the C-Cluster in Ni-Containing Carbon Monoxide Dehydrogenases Academic Article uri icon

abstract

  • The C-cluster of carbon monoxide dehydrogenase (CODH) appears to be the active site for the oxidation of CO to CO2. We have studied with EPR and Mössbauer spectroscopy the enzymes from Rhodospirillum rubrum (CODHRr; ∼8 Fe atoms and 1 Ni atom per α) and Clostridium thermoaceticum (CODHCt; ∼12 Fe atoms and 2 Ni atoms per αβ). The study of CODHRr offers two advantages. First, the enzyme lacks the A-cluster responsible for the synthase activity of CODHCt. Second, a Ni-deficient protein (Ni-deficient CODHRr) containing all Fe components of the holoenzyme can be isolated. The holoenzymes of both species can be prepared in a state for which the C-cluster exhibits the so-called gav = 1-82 EPR signal (Credl); the spectra of Ni-deficient CODHRr do not exhibit this signal. Our results are as follows: The Mössbauer data show that all iron atoms of Ni-deficient CODHRr belong to two [Fe4S4]1+/2+ clusters. The so-called B-cluster, which functions in electron transfer, is diamagnetic in the [Fe4S4]2+ state, Box, and exhibits an S = 1/2 (g = 1.94) EPR signal in the [Fe4S4]+ state, Bred. The spectroscopic properties of the B-cluster are the same in 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 incorporation 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, Cox, to the reduced state, Credl, which exhibits the gav = 1.82 EPR signal. A study of a sample poised at -300 mV shows that this signal originates from an S = 1/2 [Fe4S4]+ cluster. In this state, the cluster has a distinct subsite, ferrous component II (FCII), having ΔEQ = 2.82 mm/s and δ = 0.82 mm/s; these parameters suggest a pentacoordinate site somewhat similar to subsite Fea of the Fe4S4 cluster of active aconitase. The same values for ΔEQ and δ were observed for CODHCt. Upon addition of CN-, a potent inhibitor of CO oxidation, the ΔEQ of FCII of CODHCt changes from 2.82 to 2.53 mm/s, suggesting that CN- binds to the FCII iron. The Mössbauer studies of CODHRr showed that only ∼60% of the C-clusters were capable of attaining the Credl state; the remainder were Cox (or C*ox). For the Mössbauer sample, the EPR spin concentration of the gav = 1.82 signal was ∼65% of that determined for the g = 1.94 signal of Bred of the fully reduced sample, a result consistent with the ∼60% obtained from Mössbauer spectroscopy. When CODHRr was reduced with CO or dithionite, a fraction of the C-clusters developed a signal similar to the gav = 1.86 signal (Cred2) of CODHCt. The Mössbauer and EPR spectra of dithionite-reduced CODHRr show that a large fraction of the C-centers are in a state for which the [Fe4S4]+ cluster has S = 3/2. While the assumption of an [Fe4S4]+ cluster with an aconitase-type subsite electronically isolated from the Ni site can explain the g values of the gav = 1.82 signal and the absence of 61Ni hyperfine interactions, published resonance Raman 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 considers a weak exchange interaction (effective coupling constant j) between an S = 1 NiII site (zero-field splitting, D) and the S = 1/2 ground state of the [Fe4S4]+ cluster. This model suggests |j| < 2 cm-1, accounts for the g values of Credl, and provides an explanation for the unusual g values (gav ≈ 2.16) reported by S. W. Ragsdale and co-workers for the adducts of CODHCt with thiocyanate and cyanate. The coupling model is consistent with 61Ni EPR studies of CODH.

author list (cited authors)

  • Hu, Z., Spangler, N. J., Anderson, M. E., Xia, J., Ludden, P. W., Lindahl, P. A., & Münck, E

complete list of authors

  • Hu, Zhengguo||Spangler, Nathan J||Anderson, Mark E||Xia, Jinqiang||Ludden, Paul W||Lindahl, Paul A||Münck, Eckard

publication date

  • January 1, 1996 11:11 AM