Native mass spectrometry identifies the HybG chaperone as carrier of the Fe(CN)2CO group during maturation of E. coli [NiFe]-hydrogenase 2

Abstract

Abstract[NiFe]-hydrogenases activate dihydrogen. Like all [NiFe]-hydrogenases, hydrogenase 2 of Escherichia coli has a bimetallic NiFe(CN)2CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN)2CO group of the [NiFe]-cofactor occurs on a distinct scaffold complex comprising the HybG and HypD accessory proteins. HybG is a member of the HypC-family of chaperones that confers specificity towards immature hydrogenase catalytic subunits during transfer of the Fe(CN)2CO group. Using native mass spectrometry of an anaerobically isolated HybG–HypD complex we show that HybG carries the Fe(CN)2CO group. Our results also reveal that only HybG, but not HypD, interacts with the apo-form of the catalytic subunit. Finally, HybG was shown to have two distinct, and apparently CO2-related, covalent modifications that depended on the presence of the N-terminal cysteine residue on the protein, possibly representing intermediates during Fe(CN)2CO group biosynthesis. Together, these findings suggest that the HybG chaperone is involved in both biosynthesis and delivery of the Fe(CN)2CO group to its target protein. HybG is thus suggested to shuttle between the assembly complex and the apo-catalytic subunit. This study provides new insights into our understanding of how organometallic cofactor components are assembled on a scaffold complex and transferred to their client proteins.

Exchange of a Single Amino Acid Residue in the HybG Chaperone Allows Maturation of All H2-Activating [NiFe]-Hydrogenases in Escherichia coli

“…The supernatant obtained after centrifugation was used immediately for anaerobic protein purification. Strep-tag-II-tagged HypC and HybG were purified individually or in complex with HypD, using Strep-tactin sepharoseXT Sepharose (IBA Lifesciences, Göttingen), exactly as described ( Soboh et al, 2013 ; Arlt et al, 2021 ). His-tagged pre-HycE was purified using cobalt-charged TALON Superflow agarose (Cytiva), following the manufacturer’s instructions.…”

“…After the separation of polypeptides, they were transferred onto a nitrocellulose membrane as described ( Towbin et al, 1979 ). After blocking the membrane, interaction partners were identified by challenging with polyclonal antiserum raised against HypC, HybG, or HycE ( Pinske et al, 2015 ; Arlt et al, 2021 ). The detection was based on chemiluminescence using the Immuno-detection kit SuperSignal West Pico PLUS (Thermo Scientific, Brunswick, Germany) and an imager Amersham Imager 600 (GE Healthcare Bio-Sciences AB, Solingen, Germany).…”

“…These latter two paralogs, which are the focus of this study, are structurally and functionally related and appear to have multifarious functions, being required for both biosynthesis and delivery of the Fe(CN) 2 CO group to the Hyd large-subunit precursors ( Blokesch et al, 2001 ; Böck et al, 2006 ). They concomitantly acquire the iron ion and a CO 2 molecule, which potentially acts as a source of the CO ligand ( Soboh et al, 2013 ; Arlt et al, 2021 ); the cellular sources of these are unknown. In complex with HypD, either HypC or HybG is required to aid the synthesis of the Fe(CN) 2 CO group ( Blokesch et al, 2002 , 2004 ).…”

“…In complex with HypD, either HypC or HybG is required to aid the synthesis of the Fe(CN) 2 CO group ( Blokesch et al, 2002 , 2004 ). Once the synthesis of this group has been completed, the chaperones deliver Fe(CN) 2 CO to their designated large-subunit precursors ( Arlt et al, 2021 ). The roles of HypC and HybG are schematically summarized in Figure 1A .…”

“…A recent study has demonstrated that HybG interacts with either HypD or pre-HybC, presumably shuttling Fe(CN) 2 CO from the HypD to pre-HybC ( Arlt et al, 2021 ). These findings corroborate earlier proposals that the distinct precursor specificity shown by HybG and HypC for particular client proteins, as well as a common specificity for HypD, signifies a chaperone function for these small proteins ( Drapal and Böck, 1998 ; Wolf et al, 1998 ; Jones et al, 2004 ).…”