Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum

Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophi c growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2 .99.2) has been purified 21-fold, with a yield of 16% and a specific activi ty of 58 mnol Of CO oxidized/min/mg of protein, by a procedure that involve d differential ultracentrifugation, anion-exchange chromatography, hydropho bic interaction chromatography, and gel filtration. The purified enzyme gav e a single protein and activity band on nondenaturing polyacrylamide gel el ectrophoresis and had a molecular mass of 230,000 Da, The 230-kDa enzyme wa s composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-k Da) subunits occurring in heterohexameric (LMS), subunit composition. The 7 5-kDa polypeptide exhibited immunological cross-reactivity,vith the large s ubunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labi le S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yi elded di (carboxamidomethyl) molybdopterin cytosine dinucleotide, identifyi ng molybdopterin cytosine dinucleotide as the organic portion of the B. jap onicum CODH molybdenum cofactor. The absorption spectrum of the purified en zyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein .