Binding of Flavin Adenine Dinucleotide to Molybdenum-containing Carbon Monoxide Dehydrogenase from Oligotropha carboxidovorans

Abstract

The carbon monoxide (CO) dehydrogenase of Oligo- tropha carboxidovorans is composed of an S-selanylcys- teine-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 reconsti- tuted with the recombinant apoflavoprotein produced in Escherichia coli. The formation of the heterotrimeric complex composed of the apoflavoprotein, the molybdo- protein, and the iron-sulfur protein involves structural changes that translate into the conversion of the apofla- voprotein from non-FAD binding to FAD binding. Bind- ing of FAD to the reconstituted deflavo (LMS)2 species occurred with second-order kinetics (k?1?1350 M?1 s?1) and high affinity (Kd?1.0?10?9 M). The structure of the resulting flavo (LMS)2 species at a 2.8-Å resolution es- tablished the same fold and binding of the flavoprotein as in wild-type CO dehydrogenase, whereas the S-sela- nylcysteine 388 in the active-site loop on the molybdo- protein 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.