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Description:
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Carbon Monoxide Dehydrogenase (CODH ) , also known as Acetyl -CoA synthase (ACS ) , is one of seven known Ni containing enzymes . CODH /ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl -CoA . Recently , X -ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr ) and CODH from Carboxydothermus hydrogenoformans (CODHCh ) have been published . These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the β subunit of heterotetrameric α2β2 enzyme from Moorella thermoacetica (CODHMt ) , formerly Clostridium thermoaceticum . Neither CODHRr nor CODHCh contain an α -subunit as is found in CODHMt . The precise structure of the active site for acetyl -CoA synthase , called the A -cluster , is not known . Therefore , crystallization of the α subunit is required to solve the remaining structural features of CODH /ACS . Obtaining crystals and determining the X -ray crystal structure is a high -risk endeavor , and a second project was pursued involving the preparation , expression and analysis of various site -directed mutants of CODHMt . Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed . Visual inspection of the crystal structure of CODHRr and CODHCh , along with sequence alignments , indicates that there may be separate pathways for proton and electron transfer during catalysis . Mutants of a proposed proton transfer pathway were characterized . Four semi -conserved histidine residues were individually mutated to alanine . Two (His116Mt and His122Mt ) were essential to catalysis , while the other two (His113Mt and His119Mt ) attenuated catalysis but were not essential . Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115 . Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123 . Activity was also "rescued" by replacing His with Cys at position 116 . Mutation of conserved Lys587 near the C -cluster attenuated activity but did not eliminate it . Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala . Mutations of conserved Asn284 also attenuated activity . These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway . |