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

Oligotropha carboxidovorans is characterized by the aerobic chemolithoautotrophic utilization of CO. CO oxidation by CO dehydrogenase proceeds at a unique bimetallic [CuSMoO 2 ] 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 O 3 ] to [Mo V O 2 SH] 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.