Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri

Abstract

CO-dependent production of H2, CO2 and CH4 was detected in crude cell extracts of acetate-grown M. barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5-10 U/mg of protein). CO dehydrogenase activity was inhibited 85% by 10 .mu.M KCN and was rapidly inactivated by O2. The enzyme was nearly homogenous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (.apprx. 5%). The native purified enzyme displayed a MW of 232,000 and a 2-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain Ni by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed.