Insights into the posttranslational assembly of the Mo-, S- and Cu-containing cluster in the active site of CO dehydrogenase of Oligotropha carboxidovorans

Abstract

Oligotropha carboxidovorans is characterized by the aerobic chemolithoautotrophic utilization of CO. CO oxidation by CO dehydrogenase proceeds at a unique bimetallic [CuSMoO2] cluster which matures posttranslationally while integrated into the completely folded apoenzyme. Kanamycin insertional mutants in coxE, coxF and coxG were characterized with respect to growth, expression of CO dehydrogenase, and the type of metal center present. These data along with sequence information were taken to delineate a model of metal cluster assembly. Biosynthesis starts with the MgATP-dependent, reductive sulfuration of [Mo(VI)O3] to [Mo(V)O2SH] which entails the AAA+-ATPase chaperone CoxD. Then Mo(V) is reoxidized and Cu(1+)-ion is integrated. Copper is supplied by the soluble CoxF protein which forms a complex with the membrane-bound von Willebrand protein CoxE through RGD-integrin interactions and enables the reduction of CoxF-bound Cu(2+), employing electrons from respiration. Copper appears as Cu(2+)-phytate, is mobilized through the phytase activity of CoxF and then transferred to the CoxF putative copper-binding site. The coxG gene does not participate in the maturation of the bimetallic cluster. Mutants in coxG retained the ability to utilize CO, although at a lower growth rate. They contained a regular CO dehydrogenase with a functional catalytic site. The presence of a pleckstrin homology (PH) domain on CoxG and the observed growth rates suggest a role of the PH domain in recruiting CO dehydrogenase to the cytoplasmic membrane enabling electron transfer from the enzyme to the respiratory chain. CoxD, CoxE and CoxF combine motifs of a DEAD-box RNA helicase which would explain their mutual translation.

Fig. 1

Motifs on the amino acid sequences of the polypeptides CoxD, CoxE, CoxF and CoxG of O. carboxidovorans OM5. For bioinformatic programs employed refer to the methods section. Color coding: DEAD-box protein motifs, yellow; PH domain elements, green; VWA domain, red; histidine acid phytase motif, blue. MIDAS metal ion adhesion site, PH domain pleckstrin homology domain, VWA von willebrand factor A (integrin I), XdhC xanthine dehydrogenase C

Fig. 2

Growth experiments with strains of O. carboxidovorans in which the genes coxE (E::km) (a), coxF (F::km) (b) or coxG (G::km) (c) were inactivated by insertional mutagenesis. Bacteria were cultivated under chemolithoautotrophic conditions employing the following gas mixtures (v/v): 45 % CO, 5 % CO₂, and 50 % air (filled circle); 40 % H₂, 10 % CO₂, and 50 % air (open circle); and 30 % H₂, 5 % CO₂, 30 % CO, and 35 % air (filled square). Each data point represents the average of three optical density measurements on separate samples of the same culture. The standard deviation of such measurements was below 2.8 %. For further details see the “Materials and methods” section

Fig. 3

Translation of the proteins CoxD, CoxE and CoxF in cell-free crude extracts of wild-type O. carboxidovorans and its insertional mutants D::km, E::km or F::km. Bacteria were cultivated under chemolithoautotrophic conditions with a gas atmosphere composed of (v/v) 30 % H₂, 5 % CO₂, 30 % CO, and 35 % air. Cell-free crude extracts (200 µg protein/lane) were subjected to denaturing PAGE followed by Western blotting employing IgG antibodies directed against CoxD (a) CoxE (b) or CoxF (c)

Fig. 4

Native PAGE of CO dehydrogenases purified from O. carboxidovorans wild type or from insertional mutants. Lanes numbered 1 to 4 each received 30 μg of CO dehydrogenase from wild-type bacteria, or the mutants E::km, F::km or G::km, respectively. Gels were stained for protein with Coomassie Brilliant Blue (a) or for CO-oxidizing activity employing CO as electron donor and INT as electron acceptor (b). For experimental details refer to “Materials and methods”

Fig. 5

UV/VIS absorption spectra of CO dehydrogenases purified from wild type (a) or from the mutants in E::km (b), F::km (c) and G::km (d). Traces: a, air-oxidized; b, sparged with pure CO for 30 min; c, reduced with 650 µM dithionite under N₂ for 4 min. The insets show the visible part of the spectra at greater detail

Fig. 6

CO dehydrogenases from the mutants in coxE (a) or coxF (b) were treated with Cu¹⁺-(thiourea)₃ (filled circle) or with sodium sulfide and sodium dithionite first followed by Cu¹⁺-(thiourea)₃ (open circle). For the removal of Cu and cyanolyzable sulfur, CO dehydrogenases were incubated with potassium cyanide and then treated with sulfide/dithionite and copper as above (filled triangle). For details see the “Methods” section

Fig. 7

Mo-EPR of CO dehydrogenases (12 mg ml⁻¹; 50 mM HEPES, pH 7.2) from O. carboxidovorans wild type and the mutants E::km, F::km, or G::km (from top to bottom). The enzymes were exposed to CO (a) or treated with 5 mM sodium dithionite (b). Spectra were recorded at 120 K at a microwave frequency, modulation amplitude, and microwave power of 9.47 GHz, 1 mT and 10 mW, respectively

Fig. 8

Mo-EPR of CO dehydrogenases in the presence of l-cysteine (15 mM), sodium sulfide (15 µM) or 2-mercaptoethanol (15 mM). a Enzyme from wild-type bacteria was treated with 5 mM potassium cyanide, small molecules were removed by gel filtration, and sulfur compounds were added as indicated. b Wild-type enzyme treated with 5 mM potassium cyanide was sulfurated (5 mM sodium sulfide plus 5 mM sodium dithionite) and, after gel filtration, supplied with the indicated sulfur compounds. The enzymes from the mutant E::km (c) or F::km (d) in their as isolated state were supplied with the indicated sulfur compounds. All assays contained CO dehydrogenase (12 mg ml⁻¹) in 50 mM HEPES (pH 7.2) sparged with pure N₂. EPR spectra were recorded as detailed in the legend to Fig. 7

Fig. 9

Model showing proposed functions of the proteins CoxD, CoxE and CoxF in the assembly of the Mo- and Cu-containing cluster in the active site of folded apo-CO dehydrogenase. CoxD is an AAA+-ATPase chaperone required for the sulfuration of the trioxo-Mo ion. CoxF, which is attached to the cytoplasmic membrane through complex formation with the von Willebrand protein CoxE, introduces a Cu¹⁺-ion resulting in a catalytically competent [CuSMoO₂] center. CoxF employs suspected phytase activity for the release of Cu²⁺ attached to phytate and a putative Cu-binding motif to escort the metal ion. The respiratory electron transport system (ETS) supplies electrons for the generation of Cu¹⁺ from Cu²⁺. See the text for further explanations