Formation of a Novel Nickel-Iron Cluster and its Biological Role in Acetate Activation

Henry M Jackson Fdn For Advmt Of Military Medicine, Bethesda MD

Investigators

Abstract

Methanogens are microorganisms that produce methane gas as the end product of their metabolism, and they are critical to a wide range of anaerobic microbiological decomposition processes. Such processes include digestion in ruminants and other animals, decay in natural aquatic environments, and man-made processes such as those used for municipal and industrial waste treatment. For our society, methane, on one-hand poses potential problems as a greenhouse gas, while at the same time it also serves as an extremely useful, clean-burning fuel. Nearly two-thirds of the methane produced in Nature is derived from decomposition of acetate by methanogens, and this project is contributing new molecular details of how these organisms carry out cleavage of the carbon-carbon bond in acetate. Acetogenic and methanogenic microorganisms carry out synthesis and breakdown of large quantities of acetic acid from one-carbon precursors/products under anaerobic conditions using a highly unusual enzymatic process in which the carbon-carbon bond of acetic acid is activated. Nickel plays an indispensable role in this process, serving as a component of a unique Ni-Fe/S cluster at the active site of a 5-subunit-containing multienzyme complex (designated ACDS for acetyl-CoA decarbonylase/synthase) responsible for acetate cleavage in species of Methanosarcina. The Ni-Fe cluster is bound to the beta subunit of the complex, which also is the site for binding of substrates CoA and acetyl-CoA. Although it is now clear that Ni is an essential element for carbon-carbon bond activation, structural information on the Ni-Fe center is lacking, and little is known about the steps involved in formation and assembly of the Ni-Fe cluster. Such information is critical to develop a clear understanding of how Ni functions in the activation of acetate. The goal of this project is to identify steps in the pathway of assembly of the ACDS beta-subunit active site metal cluster by characterizing the role of the accessory protein ACDS-ORF in the process of Ni insertion into the Fe-containing apoenzyme. Incorporation of nickel is monitored by spectroscopic methods and enzymatic functional assays. Additional proteins that participate in the assembly of the beta-subunit Ni-Fe center are being sought by a combination of molecular biological two-hybrid assays and standard biochemical methods of fractionation and analysis. Detailed knowledge of how nickel functions as part of the Ni-Fe active center in the ACDS beta subunit will be obtained from experiments to characterize the coordination environment of nickel. X-ray absorption spectroscopic methods (Ni EXAFS and Ni L-edge spectroscopy) are providing the geometry, number of ligands to Ni, their chemical nature, and average bond distances. Site-directed mutagenesis experiments are being used to identify amino acid residues serving as potential ligands to Ni, and mutants will be characterized for their ability to bind Ni, undergo changes in coordination environment and to function in acetate activation. The results from this project will provide new information on the structure, function and formation of an unusual Ni-Fe/S cluster, leading to a better understanding of its biochemistry and its unique physiological role in catalyzing the activation of acetate.