Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens: CATALYTIC PROPERTIES AND EVIDENCE FOR A BINUCLEAR Ni-Ni SITE

Simonida Gencic,David A Grahame

doi:10.1074/jbc.m210484200

Abstract

The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C-C bond cleavage in methanogens growing on acetate and is also responsible for synthesis of acetyl units during growth on C-1 substrates. The ACDS beta subunit contains nickel and an Fe/S center and reacts with acetyl-CoA forming an acetyl-enzyme intermediate presumably directly involved in acetyl C-C bond activation. To investigate the role of nickel in this process two forms of the Methanosarcina thermophila beta subunit were overexpressed in anaerobically grown Escherichia coli. Both contained an Fe/S center but lacked nickel and were inactive in acetyl-enzyme formation in redox-dependent acetyltransferase assays. However, high activity developed during incubation with NiCl(2). The native and nickel-reconstituted proteins both contained iron and nickel in a 2:1 ratio, with insignificant levels of other metals, including copper. Binding of nickel elicited marked changes in the UV-visible spectrum, with intense charge transfer bands indicating multiple thiolate ligation to nickel. The kinetics of nickel incorporation matched the time course for enzyme activation. Other divalent metal ions could not substitute for nickel in yielding catalytic activity. Acetyl-CoA was formed in reactions with CoA, CO, and methylcobalamin, directly demonstrating C-C bond activation by the beta subunit in the absence of other ACDS subunits. Nickel was indispensable in this process too and was needed to form a characteristic EPR-detectable enzyme-carbonyl adduct in reactions with CO. In contrast to enzyme activation, EPR signal formation did not require addition of reducing agent, indicating indirect catalytic involvement of the paramagnetic species. Site-directed mutagenesis indicated that Cys-278 and Cys-280 coordinate nickel, with Cys-189 essential for Fe/S cluster formation. The results are consistent with an Ni(2)[Fe(4)S(4)] arrangement at the active site. A mechanism for C-C bond activation is proposed that includes a specific role for the Fe(4)S(4) center and accounts for the absolute requirement for nickel.

Highlights

The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C–C bond cleavage in methanogens growing on acetate and is responsible for synthesis of acetyl units during growth on C-1 substrates
Overexpression and Purification of the ACDS ␤ Subunit— The gene encoding the ␤ subunit of the ACDS complex from M. thermophila was overexpressed in two separate forms in E. coli
To investigate the role of the ACDS ␤ subunit in activation of the acetyl C–C bond, two forms of the protein were overexpressed in E. coli under anaerobic growth conditions and purified and characterized

Summary

Introduction

The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C–C bond cleavage in methanogens growing on acetate and is responsible for synthesis of acetyl units during growth on C-1 substrates. The ACDS ␤ subunit contains nickel and an Fe/S center and reacts with acetyl-CoA forming an acetyl-enzyme intermediate presumably directly involved in acetyl C–C bond activation. The ACDS complex catalyzes cleavage of the acetyl C–C bond using the substrates acetylCoA and tetrahydrosarcinapterin (H4SPt), a tetrahydrofolate analog which serves as methyl acceptor, and yields the products CoA, N5-methyltetrahydrosarcinapterin, CO2, and two reducing equivalents, as given in Reaction 1 [1]. Characterization of the ability of the ACDS ␤ subunit to bind acetyl-CoA and CoA, and to catalyze acetyl group transfer reactions by way of a high energy acetyl-enzyme intermediate formed on the enzyme at low redox potentials [3], and other studies [4], further implicated the ␤ subunit A center as the site where acetyl C–C bond activation takes place. A crystallographic structure of the clostridial CODH/ACS enzyme was published in which the A center was shown to contain an Fe4S4 cluster in close proximity to a binuclear metal site containing nickel and another metal ion suggested to be copper [13]

Objectives

Methods

Results

Conclusion