Metal Transfer within the Escherichia coli HypB–HypA Complex of Hydrogenase Accessory Proteins

Abstract

The maturation of [NiFe]-hydrogenase in E. coli is a complex process involving many steps and multiple accessory proteins. The two accessory proteins, HypA and HypB, interact with each other and are thought to cooperate to insert nickel into the active site of the hydrogenase-3 precursor protein. Both of these accessory proteins bind metal individually, but little is known about the metal-binding activities of the proteins once they assemble together into a functional complex. In this study, we investigate how complex formation modulates metal binding to the E. coli proteins HypA and HypB. This work lead to a re-evaluation of the HypA nickel affinity, revealing a K D on the order of 10 −8 M. HypA can efficiently remove nickel, but not zinc, from the metal-binding site in the GTPase domain of HypB, a process that is less efficient when complex formation between HypA and HypB is disrupted. Furthermore, nickel release from HypB to HypA is specifically accelerated when HypB is loaded with GDP, but not GTP. These results are consistent with the HypA-HypB complex serving as a transfer step in the relay of nickel from membrane transporter to its final destination in the hydrogenase active site, and suggest that this complex contributes to the metal fidelity of this pathway. KeywordsMetalloprotein; metal chaperone; nickel metabolism; nickel transfer; [NiFe] hydrogenase; hydrogenase maturation; HypA; HypB Many enzymes require transition metal ions at their active sites in order to function, 1, 2 and access to the appropriate metals is often vital for the survival of the organism. Strikingly, the same metals that are essential can also be toxic when distribution is not properly controlled. 3,4 An intracellular excess of one type of metal may result in competition with other metals required as cofactors for regulatory or enzymatic processes, 3,5 or catalyze the formation of † This work was supported by funding from the Canadian Institutes of Health Research as well as graduate fellowships from the Natural Sciences and Engineering Research Council of Canada (to C.D.D. and H. K.). *To whom correspondence should be addressed. Deborah B. Zamble: Tel: 416-978-3568. dzamble@chem.utoronto.ca. Author ContributionsThe manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.Supporting Information. Control experiments of mutant variants of HypB, scripts for fitting, crosslinking experiments, and predicted masses of metal-protein species are contained within the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. CIHR Author ManuscriptCIHR Author Manuscript CIHR Author Manuscript free radicals, 6 both detrimental circumstances. For this reason, organisms have intricate systems dedicated to the controlled flow of essential metals throughout the cell. 4,[7][8][9] For example, through the use of metallochaperone proteins metal ions can be directed to where they are needed, minimizing the demand for ...

Relationship between Ni(II) and Zn(II) Coordination and Nucleotide Binding by the Helicobacter pylori [NiFe]-Hydrogenase and Urease Maturation Factor HypB

“…We propose that the difference in Ni(II) affinity between the GTP-and GDP-bound states could be further magnified due to other components of the hydrogenase pathway. In support of this model, E. coli HypA stimulates nickel release from the GDP-bound, but not the GTP-bound, HypB protein (61). This mechanism of action of HypB corresponds to recent observations made with UreG (62), a closely related GTPase required for urease maturation.…”

[FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation

“…HypB's interaction with the large subunit requires the presence of HypA, providing support for HypA being the docking protein between HypB and HycE in the nickel delivery step [210,219,220]. Moreover, HypA selectively removes Ni 2+ from the GTPase domain of HypB and this metal release appears to be stimulated by GTP hydrolysis [225]. Despite the observation that Ni 2+ binding to HypB partially inhibits the GTPase activity of HypB [217], it is presumed that complex formation in vivo alleviates this retardation and promotes nickel mobilization [225].…”

“…Moreover, HypA selectively removes Ni 2+ from the GTPase domain of HypB and this metal release appears to be stimulated by GTP hydrolysis [225]. Despite the observation that Ni 2+ binding to HypB partially inhibits the GTPase activity of HypB [217], it is presumed that complex formation in vivo alleviates this retardation and promotes nickel mobilization [225]. Along these lines, it has been demonstrated that in E. coli, SlyD delivers Ni 2+ to HypB and heterodimer formation between these proteins promotes GTP hydrolysis by HypB [226].…”

CO synthesized from the central one-carbon pool as source for the iron carbonyl in O 2 -tolerant [NiFe]-hydrogenase

“…The HypF and HypE proteins deliver the CN − ligands, which are synthesized from carbamoyl phosphate (9). Incorporation of the nickel is facilitated by the HypB and HypA proteins (10). However, source and synthesis of the active site CO ligand remained elusive.…”