Characterization of canonical and noncanonical nickel metallochaperones in Methanococcus maripaludis

Abstract

Nickel is an essential trace element for many microorganisms, serving as a catalytic cofactor for enzymes involved in energy metabolism, carbon cycling, and microbial physiology. At the same time, excess intracellular nickel is toxic, requiring organisms to maintain highly regulated nickel homeostasis systems that balance nickel acquisition with controlled intracellular trafficking. Methanogenic archaea (methanogens) rely on multiple nickel-dependent enzymes that are central to methanogenesis, including [NiFe] hydrogenases. The nickel insertion step during [NiFe] hydrogenase assembly involves the nickel metallochaperones HypA and HypB. HypA is a small nickel carrier that contains an N-terminal nickel binding domain and a C-terminal structural zinc-binding site. HypB belongs to the P-loop family of GTPase with a nickel-binding site at the G-domain. In this work, we characterized the nickel metallochaperones HypA and HypB from the hydrogenotrophic methanogen Methanococcus maripaludis. MmpHypA binds either zinc or a mononuclear iron at its C-terminal metal-binding site, the latter representing an unusual metal occupancy not previously observed for HypA proteins. MmpHypB contains the canonical nickel-binding site at the G-domain that stimulates GTP hydrolysis and promotes the formation of HypA-HypB complexes. Zinc-bound MmpHypA is optimized for nickel acquisition of MmpHypB, and the nucleotide state of MmpHypB modulates the oligomeric state of HypA-HypB complexes, supporting a GTPase-driven nickel delivery mechanism. In addition to the canonical HypB protein, methanogens encode a second HypB homolog, HypB2, whose exact function remains unknown. Biochemical characterization of HypB2 proteins revealed that they form a stable complex with proteins encoded by neighboring genes. HypB2 exists predominantly as dimers and the dimeric form is stabilized by strand-swapping at the extended C-terminal region. This region also putatively coordinates a Fe-S cluster, a feature not previously associated with HypB proteins. Deletion of hypB2 does not impair growth under nickel-limiting conditions and HypB2 did not form complex(es) with HypA, suggesting that HypB2 represents a distinct member of the P-loop GTPase family that may not function as a canonical nickel metallochaperone. Instead, AP-MS experiments hint toward a role of HypB2 and its associated complex in Fe-S cofactor trafficking. Together, these findings expand current understanding of nickel trafficking in methanogens and reveal previously unrecognized diversity in the nickel metallochaperone family as well as identify a protein complex that potentially involves in Fe-S cofactor trafficking in methanogens.