Identification of a Ni- and Fe-containing cluster in Rhodospirillum rubrum carbon monoxide dehydrogenase

Carbon monoxide dehydrogenase from Clostridium thermoaceticum, with an alpha 3 beta 3 quaternary structure, was incubated with dithiothreitol and 740-78,000 equiv/alpha 3 beta 3 of sodium dodecyl sulfate (SDS), followed by electrophoresis under anaerobic native conditions. Three catalytically-active bands and four inactive bands were obtained, in proportions dependent on the amount of SDS added. The catalytically-active bands, called SM-CODH, NM-CODH, and FM-CODH, migrated slower than, similar to, and faster than native enzyme, respectively. Two-dimensional electrophoresis revealed that SM-CODH and NM-CODH contained approximately equal proportions of the alpha and beta subunits, while the beta/alpha ratio for FM-CODH was about 2.7. The four inactive bands were identified as the beta subunit, two forms of the alpha subunit (called alpha and alpha'), and a form that migrated similarly to native enzyme. Overloaded gels revealed that alpha and each active band had brown color, indicating intact Fe-S clusters in these species. Higher concentrations of SDS (> 1600 equiv/alpha 3 beta 3) and/or incubation at temperatures > 15 degrees C yielded more alpha and beta subunits at the expense of the catalytically-active bands. When incubated at 70 degrees C in 78,000 equiv/alpha 3 beta 3 of SDS, alpha quantitatively converted to alpha', suggesting that alpha' is a denatured form. FM-CODH appears to be an intermediate in the decomposition of CODH by SDS and to have either an alpha 1 beta 3 or alpha 1 beta 2 quaternary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Discussionmentioning

confidence: 99%

Decomposition of Carbon Monoxide Dehydrogenase into .alpha. Metallosubunits and a Catalytically-Active Form Consisting Primarily of .beta. Metallosubunits

Xia

Lindahl²

1995

“…It is a monomer containing 1 Ni and 7-8 Fe ions (Bonam & Ludden, 1987;Ensign et al, 1989b). The CO oxidation active site is a Ni-Fe-S cluster that exhibits an EPR signal known as signal A (gi = 2.04, g3 = 1.90, and g3 = 1.71) (Stephens et al, 1989). CO and CN™ bind directly to the Ni (Ensign et al, 1989a,b), which is bridged to an Fe-S cluster (Tanetal., 1992).…”

mentioning

confidence: 99%

Organization of Clusters and Internal Electron Pathways in CO Dehydrogenase from Clostridium thermoaceticum: Relevance to the Mechanism of Catalysis and Cyanide Inhibition

Anderson¹,

Lindahl²

1994

129

Cyanide inhibits the CO oxidation activity of carbon monoxide dehydrogenase from Clostridium thermoaceticum by binding tightly to the form of the C-cluster yielding the gav = 1.82 signal (the C1.82 form). CN- dissociates and the enzyme reactivates upon addition of CO, CO2 plus dithionite, or CS2 plus dithionite. Dithionite slows the inhibition of the enzyme by CN-, but it cannot reactivate the enzyme. This behavior is explained by assuming that binding of CO, CO2, or CS2 at a modulator site accelerates the dissociation of CN- from the C-cluster. With CN- bound at the C-cluster, dithionite, but not CO, can reduce those Fe-S clusters in the enzyme whose redox status can be monitored at 420 nm. The electron pathway used for CO oxidation appears to be as follows: C-cluster-->Fe-S Clusters-->external electron acceptors. The electron used to reduce the NiFe complex originates predominantly from the C-cluster, and this reduction is inhibited when CN- is bound at the C-cluster. The NiFe complex is reduced more slowly (in the absence of CN-) than CO is catalytically oxidized, indicating that this reduction is not part of the catalytic mechanism for CO oxidation. The form of the C-cluster yielding the g(av) = 1.86 signal (C1.86) is proposed to be two electrons more reduced than C1.82 and able to bind and reduce CO2. CO is proposed to be oxidized by C1.82. Neither CO or CN- appears to bind C1.86.

“…Evidence that nickel is a component of center C in the CODH from C. thermoaceticum has been provided by freeze-quench resonance Raman spectroscopy of the CO adduct with CODH (Qiu et al, 1995). Significant 57Fe hyperfine broadening has been observed for the CODHs from both R. rubrum (Stephens et al, 1989) and C. thermoaceticum (Lindahl et al, 1990a). Therefore, like center A , center C appears to be a Ni/Fe-S cluster.…”

mentioning

confidence: 97%

“…Although the structure of center C is not known, spectroscopic studies have given insight into its nature. The EPR spectra of a center in the CODHs of Rhodospirillum rubrum (Bonam & Ludden, 1987;Stephens et al, 1989) and Clostridium thermoaceticum (Ragsdale et al, 1982;Lindahl et al, 1990a) are highly similar indicating that center C is present in both CODHs. In contrast, center A and acetyl-CoA synthesis activity are absent from the R. rubrum enzyme.…”

mentioning

confidence: 97%

“…In contrast, center A and acetyl-CoA synthesis activity are absent from the R. rubrum enzyme. Center C of the R. rubrum enzyme was proposed to contain nickel because there is ~8 G broadening of the low-field feature of the EPR spectrum of the 61Ni-substituted enzyme (Stephens et al, 1989). Evidence that nickel is a component of center C in the CODH from C. thermoaceticum has been provided by freeze-quench resonance Raman spectroscopy of the CO adduct with CODH (Qiu et al, 1995).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Mechanism of CO oxidation by carbon monoxide dehydrogenase from Clostridium thermoaceticum and its inhibition by anions

Seravalli

Kumar

et al. 1995

Carbon monoxide dehydrogenase (CODH) performs two distinct reactions at two different metal centers. The synthesis of acetyl-CoA from a methyl group, CO, and coenzyme A occurs at center A and the oxidation of CO to CO2 occurs at center C. In the work reported here, we have studied the mechanism of CO oxidation by CODH and its inhibition by thiocyanate. Our data are consistent with a ping-pong mechanism. A scheme to explain the first half-reaction was developed that includes binding of water and CO to the oxidized form of center C, deprotonation of coordinated water to yield enzyme-bound hydroxyl, nucleophilic attack on coordinated CO by OH- to form enzyme-bound carboxyl, and deprotonation and decarboxylation to form CO2 and the reduced form of center C. In the second half-reaction, the reduced enzyme is reoxidized by an electron acceptor. CO oxidation was pH dependent. The pH dependence of kcat/Km for CO gave a single pKa of 7.7 and a maximum value at 55 degrees C and high pH of 9.1 x 10(6) M-1 s-1. The pH dependence of kcat followed a two-phase titration curve with pKa values of 7.1 and 9.5 and maximum value of kcat at 55 degrees C and high pH of 3250 s-1 (1310 mumol of CO oxidized min-1 mg-1). The pH dependencies of kcat/Km and kcat are interpreted to reflect the ionization of enzyme-bound water from binary and ternary complexes with center C. Reaction with thiocyanate, azide, or cyanate was found to cause a striking shift in the EPR spectrum of center C from gav = 1.82 (g = 2.01, 1.81, 1.65) to a two-component spectrum with gav = 2.15 (g = 2.34, 2.067, 2.03) and gav = 2.17 (g = 2.34, 2.115, 2.047). Thiocyanate acted as a mixed partial inhibitor with respect to CO. The inhibition constants were pH and temperature dependent. The pH dependencies of the inhibition constants gave pKa values of approximately 7.7. Binding of thiocyanate to the oxidized form of center C appears to be favored by a negative enthalpy that is offset by a decrease in entropy yielding a slightly unfavorable free energy of association.