MoCu CO Dehydrogenase Assembly

Abstract

AbstractAerobic chemolithoautotrophic bacteria likeOligotropha carboxidovoransbiosynthesize CO (carbon monoxide) dehydrogenases to utilize CO as energy and carbon source. The enzyme comprises an (LMS)2heterohexameric structure with each heterotrimer building up an electron transport chain. CO oxidation proceeds at a unique bimetallic [CuSMoO2] cluster within the CoxL subunit. Released electrons are subsequently transferred to the [2Fe–2S] clusters of the iron–sulfur protein CoxS. Finally, the electrons are transmitted to the FAD (flavin‐adenine‐dinucleotide) cofactor in CoxM and released to the electron transport system of the cytoplasmic membrane, generating a proton‐motive force. Using substrate analogues and performing quantum chemical modeling, different schemes for the CO oxidation reaction cycle were developed. In the first case, a thiocarbonate transition state was proposed, whereas in the second case it was assumed that binding of CO via its carbon atom to the Cu1+ion is the first step in the catalytic cycle, followed by the transfer of the equatorial oxygen‐derived ligand on the molybdenum. The metal cluster matures post‐translationally with the molybdenum cofactor already being integrated into the completely folded and fully assembled apo‐CO dehydrogenase. Furthermore, the accessory proteins CoxD, CoxE, and CoxF are required. It was suggested that biosynthesis starts with the MgATP‐dependent, reductive sulfuration of [MoVIO3] to [MoVO2SH], which involves the function of the AAA+‐ATPase chaperone CoxD. After reoxidation of the molybdenum, Cu1+is inserted. The latter is supplied by soluble CoxF, which forms a complex with the membrane‐bound von Willebrand protein CoxE. Within this complex, Cu2+is reduced to Cu1+by electrons from respiration. Copper appears as Cu2+‐phytate, is mobilized through the phosphatase/phytase activity of CoxF, and is subsequently transferred to the CoxF copper‐binding site. Knowledge of the molybdenum and copper oxidation states during active‐site cluster assembly also enables the chemical reconstitution of the active site. CoxD, CoxE, and CoxF combine motifs of a DEAD‐box RNA helicase, which leads to a mutual translation of the polypeptides. The presence of a pleckstrin homology (PH) domain on CoxG suggests a role in recruiting CO dehydrogenase to the cytoplasmic membrane enabling electron transfer from the enzyme to the respiratory chain.