Resolution of Component Proteins in an Enzyme Complex from Methanosarcina thermophila Catalyzing the Synthesis or Cleavage of Acetyl-CoA

• Published in: Proceedings of the National Academy of Sciences - PNAS 1991-04, Vol.88 (8), p.3272-3276
• Main Authors: Abbanat, Darren R., Ferry, James G.
• Format: Article
• Language: English
• Subjects: 550700 - Microbiology; Acetates; acetyl-CoA; AMINES; Analytical, structural and metabolic biochemistry; BACTERIA; BASIC BIOLOGICAL SCIENCES; Biochemistry; Biological and medical sciences; CARBON COMPOUNDS; CARBON MONOXIDE; CARBON OXIDES; CHALCOGENIDES; CHEMICAL REACTIONS; CHROMATOGRAPHY; Cobalt; COENZYMES; cofactors; Corrinoids; DYES; Electrons; ENZYMES; Enzymes and enzyme inhibitors; FERREDOXIN; Ferredoxins; Ferries; Fundamental and applied biological sciences. Psychology; ION EXCHANGE CHROMATOGRAPHY; METABOLISM; METALLOPROTEINS; METHANOGENIC BACTERIA; METHYL VIOLET; MICROORGANISMS; Miscellaneous; MOLECULAR STRUCTURE; ORGANIC COMPOUNDS; OXIDATION; OXIDES; OXYGEN COMPOUNDS; PROTEINS; SEPARATION PROCESSES; SPECTRA; Sulfides; Sulfur; TRIPHENYLMETHANE DYES; ULTRAVIOLET SPECTRA
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.8.3272

An enzyme complex was isolated from acetate-grown Methanosarcina thermophila that oxidized CO and catalyzed the synthesis or cleavage of acetyl-CoA. The complex consisted of five subunits (α1β1γ1δ1ε1) of 89, 71, 60, 58, and 19 kDa. The complex contained nickel, iron, acid-labile sulfide, and cobalt in a corrinoid cofactor. Two components were resolved by anion-exchange chromatography of the complex in the presence of dodecyltrimethylammonium bromide and Triton X-100: a 200-kDa nickel/iron-sulfur protein with the 89- and 19-kDa (α2εx) subunits and a 100-kDa corrinoid/iron-sulfur protein with the 60- and 58-kDa subunits (γ1δ1). The nickel/iron-sulfur component contained 0.21 Ni, 2.7 Zn, 7.7 Fe, and 13.2 acid-labile sulfide (per α1ε1). The corrinoid/iron-sulfur component contained 0.7 Co, 0.7 factor III {Coα-[α-(5-hydroxybenzimidazolyl)]-Coβ-cyanocobamide}, 3.0 Fe, and 2.9 acid-labile sulfide (γ1δ1). Both components contained iron-sulfur centers. The nickel/iron-sulfur component oxidized CO and reduced methyl viologen or a ferredoxin isolated from M. thermophila. The nickel/iron-sulfur component also oxidized CO and transferred electrons to the corrinoid/iron-sulfur component, reducing the iron-sulfur and Co centers. UV-visible spectroscopy indicated that the reduced corrinoid/iron-sulfur component could be methylated with CH3I. The results suggest that the enzyme complex from M. thermophila contained at least two enzyme components, each with a specific function. The properties of the component enzymes support a mechanism proposed for acetyl-CoA synthesis (or cleavage) by the enzyme complex.