Support for Nickel as the Labile Metal in the A‐center of the M. Barkeri Acetyl‐CoA Decarbonylase/Synthase Complex

Abstract

AbstractThe α8β8γ8δ8ϵ8 acetyl‐CoA decarbonylase synthase (ACDS) complex catalyzes the first step in the conversion of acetate to methane. The critical C‐C bond cleavage of the acetyl group is mediated by the A‐center, aNi‐X‐Fe4S4 cluster that has been localized to the β‐subunit. To elucidate the identity of the labile metal X in the A‐center of purified enzyme, the β‐subunit of the M. barkeri ACDS complex was isolated and studied. Metal analysis revealed the presence of both nickel (1.9) and copper (0.2) per subunit indicating a mixture at the metal site. Treatment of the β‐subunit with the Cu(I) specific chelator, bathocuproine disulfonate (BCS), allowed for the complete removal of the copper in the sample as confirmed by fluorescence quenching of the BCS chelator and subsequent metal analysis. The BCS‐treated β‐subunit was found to have nearly identical activity to the as‐isolated enzyme; importantly, addition of Ni(II) to this BCS‐treated β‐subunit resulted in a 24% increase in acetyltransferase activity over that of the isolated enzyme consistent with activation of the A‐center by incorporation of Ni(II) into sites previously occupied by Cu. These results suggest that while Cu can occupy the labile metal site, it is the Ni‐bound form that is active ‐ providing additional support that the A‐center of the methanogenic ACS consists of aNi‐Ni‐Fe4S4 cluster. Perhaps equally important, these studies demonstrate that BCS/Ni(II) treatment provides a means of curing samples from Cu inactivation ‐ a key step towards obtaining the uniform A‐center needed for detailed structural and mechanistic studies.