The Mechanism of Bacterial Cu+-ATPases. Distinct Efflux Rates Adapted to Different Function

Abstract

Chemistry and Biochemistry Department, Worcester Polytechnic Institute, Worcester, MA, USA Copper plays important physiological roles as a protein co-factor. In bacteria, cytoplasmic copper homeostasis is controlled by P1B-1-type Cu+-ATPases. These highly homologous transporters share a common structure and the classical Albers-Post (E1/E2) mechanism. It is accepted that most of these enzymes drive cytoplasmic metal efflux and consequently confer Cu+ tolerance. Other members of this subfamily appear required for cuproprotein assembly. Early studies using gene deletion, phenotypical characterization and functional complementation, have suggested that these are in fact Cu+ importers. To explain this phenotypical observation within the mechanistic constrains of the transport cycle of P-type ATPases, we have studied the Cu+ transport by three proposed Cu+ importers: P. aeruginosa CopA2, E. hirae CopA and Synechocystis PCC6803 CtaA. These were expressed in an E. coli strain lacking its endogenous Cu+-ATPase (DC194), functional complementation was tested, and 64Cu+ transport into everted vesicles measured. These experiments show that all Cu+- ATPases drive Cu+ efflux although with quite different kinetics. ATPases involved in cuproprotein assembly have much slower transport rates and high apparent metal affinities. These characteristics explain phenotypes observed upon mutation of the coding genes.