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Like all [NiFe]-hydrogenases, hydrogenase 2 of\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    has a bimetallic NiFe(CN)\n                    <jats:sub>2<\/jats:sub>\n                    CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN)\n                    <jats:sub>2<\/jats:sub>\n                    CO 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)\n                    <jats:sub>2<\/jats:sub>\n                    CO group. Using native mass spectrometry of an anaerobically isolated HybG\u2013HypD complex we show that HybG carries the Fe(CN)\n                    <jats:sub>2<\/jats:sub>\n                    CO 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 CO\n                    <jats:sub>2<\/jats:sub>\n                    -related, covalent modifications that depended on the presence\u00a0of the\n                    <jats:italic>N<\/jats:italic>\n                    -terminal cysteine residue on the protein, possibly representing intermediates during Fe(CN)\n                    <jats:sub>2<\/jats:sub>\n                    CO group biosynthesis. Together, these findings suggest that the HybG chaperone is involved in both biosynthesis and delivery of the Fe(CN)\n                    <jats:sub>2<\/jats:sub>\n                    CO 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.\n                  <\/jats:p>","DOI":"10.1038\/s41598-021-03900-w","type":"journal-article","created":{"date-parts":[[2021,12,21]],"date-time":"2021-12-21T06:03:53Z","timestamp":1640066633000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Native mass spectrometry identifies the HybG chaperone as carrier of the Fe(CN)2CO group during maturation of E. coli [NiFe]-hydrogenase 2"],"prefix":"10.1038","volume":"11","author":[{"given":"Christian","family":"Arlt","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kerstin","family":"Nutschan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Haase","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Ihling","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dirk","family":"T\u00e4nzler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1521-4899","authenticated-orcid":false,"given":"Andrea","family":"Sinz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0862-2683","authenticated-orcid":false,"given":"R. 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