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Journal of Bacteriology, September 2001, p. 5134-5144, Vol. 183, No. 17
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.17.5134-5144.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Two Membrane-Associated NiFeS-Carbon Monoxide Dehydrogenases from the Anaerobic Carbon-Monoxide-Utilizing Eubacterium Carboxydothermus hydrogenoformans

Vitali Svetlitchnyi,1 Christine Peschel,1 Georg Acker,2 and Ortwin Meyer1,3,*

Lehrstuhl für Mikrobiologie,1 Fachgruppe Biologie-Elektronenmikroskopie,2 and Bayreuther Zentrum für Molekulare Biowissenschaften,3 Universität Bayreuth, D-95440 Bayreuth, Bavaria, Germany

Received 22 January 2001/Accepted 4 June 2001

Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium Carboxydothermus hydrogenoformans. Both enzymes differ in their subunit molecular masses, N-terminal sequences, peptide maps, and immunological reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in association with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO + H2O right-arrow CO2 + 2 e- + 2 H+. Oxidized viologen dyes are effective electron acceptors. The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) µmol of CO oxidized min-1 mg-1 of protein (methyl viologen, pH 8.0, 70°C). The two enzymes oxidize CO very efficiently, as indicated by kcat/Km values at 70°C of 1.3 · 109 M-1 CO s-1 (CODH I) and 1.7 · 109 M-1 CO s-1 (CODH II). The apparent Km values at pH 8.0 and 70°C are 30 and 18 µM CO for CODH I and CODH II, respectively. Acetyl coenzyme A synthase activity is not associated with the enzymes. CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers containing 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, respectively. Electron paramagnetic resonance (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.

* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie, Universität Bayreuth, D-95440 Bayreuth, Bavaria, Germany. Phone: 49 (921) 552729. Fax: 49 (921) 552727. E-mail: ortwin.meyer{at}uni-bayreuth.de.

Journal of Bacteriology, September 2001, p. 5134-5144, Vol. 183, No. 17
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.17.5134-5144.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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