Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans - PubMed (original) (raw)

Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans

V Svetlitchnyi et al. J Bacteriol. 2001 Sep.

Abstract

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 + H(2)O --> CO(2) + 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) micromol of CO oxidized min(-1) mg(-1) of protein (methyl viologen, pH 8.0, 70 degrees C). The two enzymes oxidize CO very efficiently, as indicated by k(cat)/K(m) values at 70 degrees C of 1.3. 10(9) M(-1) CO s(-1) (CODH I) and 1.7. 10(9) M(-1) CO s(-1) (CODH II). The apparent K(m) values at pH 8.0 and 70 degrees C are 30 and 18 microM 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.

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Figures

FIG. 1

Analysis of CODH I and CODH II by PAGE. CODH I and CODH II were subjected to native PAGE (A), SDS-PAGE (B), peptide mapping (C), and Western blot analysis (D). (A) Native PAGE. Lane 1, molecular mass markers; lane 2, 10 μg of CODH I; lane 3, 10 μg of CODH II. (B) SDS-PAGE. Lane 4, 5 μg of CODH I; lane 5, 5 μg of CODH II; lane 6, molecular mass markers. (C) Peptide mapping of 10 μg of CODH I or CODH II by limited proteolysis with pepsin or α-chymotrypsin for 30 min. Lane 7, pepsin digest of CODH I; lane 8, pepsin digest of CODH II; lane 9, α-chymotrypsin digest of CODH I; lane 10, α-chymotrypsin digest of CODH II (the number 49 refers to the 49-kDa peptide which has been sequenced). (D) Western blot analysis with purified polyclonal IgG antibodies directed against CODH I or CODH II (10 μg of each). Lane 11, CODH I and antibodies directed against CODH I; lane 12, CODH II and antibodies directed against CODH I; lane 13, CODH I and antibodies directed against CODH II; lane 14, CODH II and antibodies directed against CODH II.

FIG. 2

UV-visible absorption spectra of CODH I (A) and CODH II (B). The enzymes (0.2 mg ml−1) were in 50 mM Tris-HCl (pH 8.0). Conditions for each curve: a, under N2 as isolated, dithionite was removed by gel filtration; b, oxidized with air; c, reduced with pure CO; d, reduced with 2 mM dithionite under N2. Insets: difference spectra of condition a minus condition c.

FIG. 3

EPR spectra of CODH I (A) and CODH II (B). CODH I (3.8 mg ml−1) or CODH II (3.1 mg ml−1) were in 50 mM Tris-HCl (pH 8.0). Traces: (a and e) as-isolated freshly prepared enzyme was frozen under N2; (b and f) air-oxidized as-isolated enzyme was kept under air for 12 h at 4°C; (c and g) CO-reduced as-isolated enzyme was kept under pure CO for 60 min at 50°C; (d and h) dithionite-reduced as-isolated enzyme was treated with 4 mM dithionite under pure N2. The relevant g values are indicated in the spectra. General conditions: 10 mW microwave power; 10 G modulation amplitude; 9.47 GHz microwave frequency; temperatures (in degrees K) as indicated.

FIG. 4

Localization of CODH I and CODH II on immunogold-labeled ultrathin sections of CO-grown C. hydrogenoformans. (A) Ultrastructure of the cell envelope after low-temperature embedding. CM, cytoplasmic membrane; S, surface layer; WL1, first wall layer; WL2, second wall layer; WL3, third wall layer. (B) Control labeling was with preimmune serum and gold-labeled secondary IgG antibodies. IgG antibodies directed against CODH I or CODH II were absent. (C and F) Longitudinal and cross-sections after labeling with IgG antibodies directed against CODH I and the gold-labeled secondary IgG antibodies. (D and G) Treated as for panels C and F, except that IgG antibodies directed against CODH II were used. (E) Tangential section parallel to the long cell axis; all other conditions were as for panels C and F. (H) Tangential section through a cell pole; all other conditions were as for panels C and F. Bar, 0.1 μm.

FIG. 5

Hypothetical scheme showing the function of the two CODHs in C. hydrogenoformans. CODH I is involved in energy generation and CODH II serves anabolic functions. For details refer to the text. The scheme is not intended to give correct stoichiometries. Abbreviations: B, ferredoxin-like protein B; H2ase, membrane-bound [NiFe] hydrogenase.

References

1. Acetarin J-D, Carlemalm E, Villiger W. Development of new Lowicryl resins for embedding biological specimens at even lower temperature. J Microsc. 1986;143:81–88. - PubMed
1. Acker G. Immunoelectron microscopy of surface antigens (polysaccharides) of gram-negative bacteria using pre- and post-embedding techniques. In: Mayer F, editor. Electron microscopy in microbiology. Methods in microbiology. Vol. 20. London, England: Academic Press Ltd.; 1988. pp. 147–174.
1. Albracht S P. The use of electron-paramagnetic-resonance spectroscopy to establish the properties of nickel and the iron-sulfur cluster in hydrogenase from Chromatium vinosum. Biochem Soc Trans. 1985;13:582–585. - PubMed
1. Albracht S P, Hedderich R. Learning from hydrogenases: location of a proton pump and of a second FMN in bovine NADH-uniquinone oxidoreductase (Complex I) FEBS Lett. 2000;485:1–6. - PubMed
1. Beisenherz G, Bolze H J, Bücher T, Czok R, Garbade K H, Meyer-Arendt E, Pfleiderer G. Diphosphofructose-Aldolase, Phosphoglyceraldehyd-Dehydrogenase, Milchsäure-Dehydrogenase, Glycerophosphat-Dehydrogenase and Pyruvat-Kinase aus Kaninchenmuskulatur in einem Arbeitsgang. Z Naturforsch B. 1953;8:555–577.

Two membrane-associated NiFeS-carbon monoxide dehydrogenases from the anaerobic carbon-monoxide-utilizing eubacterium Carboxydothermus hydrogenoformans - PubMed (original) (raw)