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To identify the metabolic origins of these ligands, the regulatory [NiFe] hydrogenase in conjunction with the indigenous Hyp maturation proteins of <jats:italic>Ralstonia eutropha<\/jats:italic> H16 were heterologously overproduced in <jats:italic>E. coli<\/jats:italic> grown in the presence of <jats:sc>l<\/jats:sc>\u2010[ureido\u2010<jats:sup>13<\/jats:sup>C] citrulline and NaH<jats:sup>13<\/jats:sup>CO<jats:sub>3<\/jats:sub>. Infrared spectroscopy of purified hydrogenase provided direct evidence that only the cyanide ligands, but not the CO ligand, originate from CO<jats:sub>2<\/jats:sub> and carbamoylphosphate. Incorporation of label from <jats:sup>13<\/jats:sup>CO exclusively into the carbonyl ligand indicates that free CO is a possible precursor in carbonyl ligand biosynthesis.<\/jats:p>","DOI":"10.1016\/j.febslet.2007.06.027","type":"journal-article","created":{"date-parts":[[2007,6,22]],"date-time":"2007-06-22T11:22:43Z","timestamp":1182511363000},"page":"3322-3326","source":"Crossref","is-referenced-by-count":53,"title":["Carbamoylphosphate serves as the source of CN<sup>\u2212<\/sup>, but not of the intrinsic CO in the active site of the regulatory [NiFe]\u2010hydrogenase from <i>Ralstonia eutropha<\/i>"],"prefix":"10.1002","volume":"581","author":[{"given":"Oliver","family":"Lenz","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ingo","family":"Zebger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Josta","family":"Hamann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Hildebrandt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"B\u00e4rbel","family":"Friedrich","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2007,6,21]]},"reference":[{"key":"e_1_2_6_2_1","unstructured":"R. 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