title: Binding of flavin adenine dinucleotide to molybdenum-containing carbon monoxide dehydrogenase from Oligotropha carboxidovorans. Structural and functional analysis of a carbon monoxide dehydrogenase species in which the native flavoprotein has been replaced by its recombinant counterpart produced in Escherichia coli.
abstract: The carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is composed of an S-selanylcysteine-containing 88. 7-kDa molybdoprotein (L), a 17.8-kDa iron-sulfur protein (S), and a 30.2-kDa flavoprotein (M) in a (LMS)(2) subunit structure. The flavoprotein could be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate. The resulting M(LS)(2)- or (LS)(2)-structured CO dehydrogenase species could be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli. The formation of the heterotrimeric complex composed of the apoflavoprotein, the molybdoprotein, and the iron-sulfur protein involves structural changes that translate into the conversion of the apoflavoprotein from non-FAD binding to FAD binding. Binding of FAD to the reconstituted deflavo (LMS)(2) species occurred with second-order kinetics (k(+1) = 1350 M(-1) s(-1)) and high affinity (K(d) = 1.0 x 10(-9) M). The structure of the resulting flavo (LMS)(2) species at a 2.8-A resolution established the same fold and binding of the flavoprotein as in wild-type CO dehydrogenase, whereas the S-selanylcysteine 388 in the active-site loop on the molybdoprotein was disordered. In addition, the structural changes related to heterotrimeric complex formation or FAD binding were transmitted to the iron-sulfur protein and could be monitored by EPR. The type II 2Fe:2S center was identified in the N-terminal domain and the type I center in the C-terminal domain of the iron-sulfur protein.
authors.affiliation: Lehrstuhl für Mikrobiologie, Universität Bayreuth, D-95440 Bayreuth, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Gremer
authors.forename: S
authors.initials: S
authors.lastname: Kellner
authors.forename: H
authors.initials: H
authors.lastname: Dobbek
authors.forename: R
authors.initials: R
authors.lastname: Huber
authors.forename: O
authors.initials: O
authors.lastname: Meyer
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005470:Fluorometry; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008982:Molybdenum / Q000737:chemistry / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D008982:Molybdenum; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase