Evidence for a Proton Transfer Network and a Required Persulfide-Bond-Forming Cysteine Residue in Ni-Containing Carbon Monoxide Dehydrogenases

Abstract

Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO 2 at a nickel-iron-sulfur active-site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semi-conserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 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. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no EPR signals originating from the C-cluster.Electronic absorption and metal analysis suggests that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity. Ni-containing carbon monoxide dehydrogenases (CODH's) are found in methanogenic archaea, acetogenic bacteria, and CO-utilizing bacteria, where they play critical roles in C 1 - In this study, we tested these hypotheses using site-directed mutagenesis, enzyme activity assays and EPR spectroscopy. We also examined the effect of mutating the cysteine residue involved in persulfide bond formation. Our results indicate that the persulfide-associated cysteine residue is required for catalysis and possibly for C-cluster assembly or stability. Our evidence also suggests that the 4 His residues as well as Lys587 and Asn284 are indeed used as a catalytic base and a proton relay network. Experimental ProceduresOligonucleotides used to construct mutants were synthesized in the Gene TechnologiesLaboratory at Texas A&M University. Mutants were constructed using the QuikChange sitedirected mutagenesis method from Stratagene, using plasmid pTM02, which contains the genes acsA and acsB, as the template (10). Double mutants were constructed using one oligonucleotide containing both mutations, except for Asn284Ala:His119Ala and Lys587Ala:His113Ala, for which two different oligonucleotides were used in two different PCR reactions. All mutant plasmids were produced using an MJ Research Minicycler PCR machine. Mutants were transformed, expressed, harvested, and purified as described (10). ...

Structure of the α 2 ε 2 Ni-dependent CO dehydrogenase component of the Methanosarcina barkeri acetyl-CoA decarbonylase/synthase complex

“…Cysteine ligands Cys-323, -523, -552, and -587 bind Fe2, Fe3, Fe4, and Ni1 atoms of the NiFe 4 S 4 psuedocubane, respectively, whereas Cys-278 and His-250 serve as protein ligands to the exogenous iron (Fe1), also known as the FCII iron. A relatively conserved exogenous ligand, Cys-277, implicated in persulfide bridge formation (27), is located near but oriented away from the S3 sulfur of the C cluster.…”

Acetyl‐coenzyme A Synthases and Nickel‐Containing Carbon Monoxide Dehydrogenases

“…Reaction (1) also involves the transfer of protons into/out of the C-cluster. As fi rst suggested by Doukov et al [4] and later confi rmed by the site-directed mutagenesis study of Kim et al [22] there is a network of conserved residues that allows protons to migrate between the C-cluster and external proton acceptors/ donors in solution. For most enzymes, the residues involved include 4 His and perhaps Lys and an Asn (see Table 2).…”

“…The idea would be that this bond is reduced in C red2 and oxidized in C red1 . However, the C-cluster appears to be absent in a mutant enzyme lacking this cysteine, suggesting that this persulfi de plays a role in either assembling or stabilizing the cluster [22].…”

Interaction of Potassium Cyanide with the [Ni-4Fe-5S] Active Site Cluster of CO Dehydrogenase from Carboxydothermus hydrogenoformans

“…4D). The proton of the thiol group is subsequently transmitted via His-261 to His-93 and Lys-563 of the assumed proton transfer chain (33), whereas the sulfide stays bound to Fe1 (Fig. 4E).…”