ABSTRACT
Crystal structures of carbon monoxide dehydrogenase (CODH), a seleno-molybdo-iron-sulfur flavoprotein from the aerobic carbon monoxide utilizing carboxidotrophic eubacterium Hydrogenophaga pseudoflava, have been determined from the enzyme synthesized at high (Mo(plus) CODH) and low intracellular molybdenum content (Mo(minus) CODH) at 2.25 A and 2.35 A resolution, respectively. The structures were solved by Patterson search methods utilizing the enzyme from Oligotropha carboxidovorans as the initial model. The CODHs from both sources are structurally very much conserved and show the same overall fold, architecture and arrangements of the molybdopterin-cytosine dinucleotide-type of molybdenum cofactor, the type I and type II [2Fe-2S] clusters and the flavin-adenine dinucleotide. Unlike the CODH from O. carboxidovorans, the enzyme from H. pseudoflava reveals a unique post-translationally modified C(gamma)-hydroxy-Arg384 residue which precedes the catalytically essential S-selanyl-Cys385 in the active-site loop. In addition, the Trp193 which shields the isoalloxazine ring of the flavin-adenine dinucleotide in the M subunit of the H. pseudoflava CODH is a Tyr193 in the O. carboxidovorans CODH. The hydrogen bonding interaction pattern of the molybdenum cofactor involves 27 hydrogen bonds with the surrounding protein. Of these, eight are with the cytosine moiety, eight with the pyrophosphate, six with the pyranopterin, and five with the ligands of the Mo ion. The structure of the catalytically inactive Mo(minus) CODH indicates that an intracellular Mo-deficiency affects exclusively the active site of the enzyme as an incomplete non-functional molybdenum cofactor was synthesized. The 5'-CDP residue was present in Mo(minus) CODH, whereas the Mo-pyranopterin moiety was absent. In Mo(plus) CODH the selenium faces the Mo ion and flips away from the Mo site in Mo(minus) CODH. The different side-chain conformations of the active-site residues S-selanyl-Cys385 and Glu757 in Mo(plus) and Mo(minus) CODH indicate a side-chain flexibility and a function of the Mo ion in the proper orientation of both residues.
Subject(s)
Aldehyde Oxidoreductases/chemistry , Aldehyde Oxidoreductases/metabolism , Betaproteobacteria/enzymology , Coenzymes/metabolism , Molybdenum/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Aldehyde Oxidoreductases/biosynthesis , Amino Acid Sequence , Betaproteobacteria/metabolism , Binding Sites , Coenzymes/deficiency , Conserved Sequence , Crystallography, X-Ray , Enzyme Stability , Flavin-Adenine Dinucleotide/metabolism , Hydrogen Bonding , Iron-Sulfur Proteins/biosynthesis , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Ligands , Metalloproteins/biosynthesis , Metalloproteins/chemistry , Metalloproteins/metabolism , Models, Molecular , Molecular Sequence Data , Molybdenum/deficiency , Multienzyme Complexes/biosynthesis , Protein Structure, Secondary , Selenium/metabolism , Sequence AlignmentABSTRACT
The molybdenum-containing iron-sulfur flavoprotein CO dehydrogenase is expressed in a catalytically fully competent form during heterotrophic growth of the aerobic bacterium Hydrogenophaga pseudoflava with pyruvate plus CO. We have adopted these conditions for studying the effect of molybdate (Mo) and tungstate (W) on the biosynthesis of CO dehydrogenase and its molybdopterin (MPT) cytosine-dinucleotide-(MCD)-type molybdenum cofactor. W was taken up by the Mo transport system and, therefore, interfered with Mo transport in an antagonistic way. Depletion of Mo from the growth medium as well as inclusion of excess W both resulted in the absence of intracellular Mo and led to the biosynthesis of CO dehydrogenase species of proper L2M2S2 subunit structure that carried the two 2Fe:2S type-I and type-II centers and two FAD molecules. EPR, ultraviolet/visible and CD spectroscopies established the full functionality of the cofactors. Due to the absence of the Mo-MCD cofactor, the enzyme species were catalytically inactive. Unexpectedly, the following cytidine nucleotides were present in inactive CO dehydrogenase: CDP, dCDP, CMP, dCMP, CTP or dCTP. The sum of cytidine nucleotides was two/mol enzyme. The binding specificities of inactive CO dehydrogenase for cytidine nucleotides (oxy > deoxy; diphosphate > monophosphate > triphosphate), and the absence of MPT suggest that, in active CO dehydrogenase, the cytidine diphosphate moiety of Mo-MCD provides the strongest interactions with the protein and determines the specificity for the type of nucleotide. In H. pseudoflava, the biosynthesis of MPT (identified as form A) was independent of Mo. Mo was, however, strictly required for the conversion of MPT to MCD (identified as form-A-CMP) as well as the insertion of Mo-MCD into CO dehydrogenase. These data support a model for the involvement of Mo in the biosynthesis of the Mo-MCD cofactor and of fully functional CO dehydrogenase in which the synthesis and insertion of Mo-MCD require Mo, and protein synthesis including integration of the FeS-centers and FAD are independent of Mo.
