Interaction between Hydrogenase Maturation Factors HypA and HypB Is Required for [NiFe]-Hydrogenase Maturation Kwok-Ho Chan, Ka-Man Lee, Kam-Bo Wong* School of Life Sciences, Centre for Protein Science and Crystallography, The Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region, People’s Republic of China Abstract The active site of [NiFe]-hydrogenase contains nickel and iron coordinated by cysteine residues, cyanide and carbon monoxide. Metal chaperone proteins HypA and HypB are required for the nickel insertion step of [NiFe]-hydrogenase maturation. How HypA and HypB work together to deliver nickel to the catalytic core remains elusive. Here we demonstrated that HypA and HypB from Archaeoglobus fulgidus form 1:1 heterodimer in solution and HypA does not interact with HypB dimer preloaded with GMPPNP and Ni. Based on the crystal structure of A. fulgidus HypB, mutants were designed to map the HypA binding site on HypB. Our results showed that two conserved residues, Tyr-4 and Leu-6, of A. fulgidus HypB are required for the interaction with HypA. Consistent with this observation, we demonstrated that the corresponding residues, Leu-78 and Val-80, located at the N-terminus of the GTPase domain of Escherichia coli HypB were required for HypA/HypB interaction. We further showed that L78A and V80A mutants of HypB failed to reactivate hydrogenase in an E. coli DhypB strain. Our results suggest that the formation of the HypA/HypB complex is essential to the maturation process of hydrogenase. The HypA binding site is in proximity to the metal binding site of HypB, suggesting that the HypA/HypB interaction may facilitate nickel transfer between the two proteins. Citation: Chan K-H, Lee K-M, Wong K-B (2012) Interaction between Hydrogenase Maturation Factors HypA and HypB Is Required for [NiFe]-Hydrogenase Maturation. PLoS ONE 7(2): e32592. doi:10.1371/journal.pone.0032592 Editor: Andy T. Y. Lau, Shantou University Medical College, China Received August 22, 2011; Accepted January 27, 2012; Published February 27, 2012 Copyright: 2012 Chan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by a General Research Fund (CUHK 4610/06M) and a Collaborative Research Fund (HKU1/07C) from Research Grants Council of Hong Kong (http://www.ugc.edu.hk/eng/rgc/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kbwong@cuhk.edu.hk Introduction Hydrogenase maturation factors HypA and HypB are involved in the delivery of nickel into the catalytic core of [NiFe]- hydrogenase [1,2,3,4,5,6,7]. Nickel and iron ions in the catalytic core of [NiFe]-hydrogenase are coordinated by two CN and one CO molecules and a network of cysteine residues [8]. Formation of the catalytic core of [NiFe]-hydrogenase requires accessory proteins encoded by genes designated hypA to hypF in the hyp operon. The Fe center is assembled before the insertion of nickel [9,10,11]. The hydrogenase maturation factors HypC, HypD, HypE and HypF are responsible for the synthesis and transfer of the Fe(CN)2 complex to the hydrogenase precursor [12,13,14,15]. The mechanism of CO ligand synthesis is unclear [16,17,18]. After delivery of the Fe(CN)2CO complex to the hydrogenase, nickel is transferred to the precursor with the help of HypA and HypB. The last step of catalytic core assembly involves proteolytic cleavage of the C-terminal tail of the large subunit after nickel insertion [19]. Disruption of hypA or hypB genes in various microorganisms resulted in hydrogenase deficiency that can be partially overcome by supplementation of nickel in the growing media [1,2,3,4,5,6,7]. HypA is a metallochaperone that binds both zinc and nickel. The zinc ion is bound with nanomolar affinity by two conserved CxxC motifs in HypA and it stabilizes the loop structure in HypA [20,21,22,23]. HypA binds nickel via the conserved residues His-2 and Glu-3 [22,23]. Mutagenesis study suggests that nickel binding via these two residues is essential for hydrogenase maturation [20]. HypB is a metal-binding GTPase. Mutagenesis study showed that both metal binding [24,25] and GTP hydrolysis activity [3,20,26] are required for hydrogenase maturation. How HypA and HypB facilitate nickel insertion into hydrog- enase is unclear. Heterodimers of HypA/HypB from Escherichia coli and Helicobacter pylori were detected in cross-linking experiments and these complex formation were independent of nickel and GTP [20,21]. Although HypA and HypB are known to be interacting partners, the significance of such interaction to hydrogenase maturation has never been addressed. Archaeoglobus fulgidus is a hyperthermophilic archeae that can grow organoheterotrophi- cally, using a variety of carbon-based energy sources, and lithoautotrophically on hydrogen, thiosulphate and carbon dioxide. We have previously determined the crystal structure of HypB from A. fulgidus (AfHypB; PDB code: 2WSM). Based on scanning mutagenesis, we identified that the unstructured N- terminal region of the HypB GTPase domain is responsible for HypA binding. We further demonstrated that amino acid substitutions of HypB which disrupt HypA/HypB interaction also abolished in vivo hydrogenase maturation. These observations suggested that interaction between HypA and HypB is essential for nickel insertion to hydrogenase precursor to produce mature PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32592