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The recent recognition of the widespread distribution of three-domain CuNiRs where cognate redox partners are fused to the core NiR enzyme offered the possibility of studying coordinated events (\n                    <jats:italic>e.g.<\/jats:italic>\n                    proton-coupled electron transfer) in a conformationally stable donor\u2013acceptor complex. The C-terminal cytochrome\n                    <jats:italic>c<\/jats:italic>\n                    tethered domain of the CuNiR from\n                    <jats:italic>Ralstonia pickettii<\/jats:italic>\n                    (\n                    <jats:italic>Rp<\/jats:italic>\n                    NiR) has been well studied. Reverse engineering of\n                    <jats:italic>Rp<\/jats:italic>\n                    NiR undertaken to remove the cognate partner domain showed that the presence of the additional domain resulted in significant differences in the apparent\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>m<\/jats:sub>\n                    for nitrite and the reduction potentials of the Cu centres when compared with the core enzyme. The oxidation state of the haem centre and the position of the tethering linker have also been shown to control access of substrate to the active site. A key feature of this control is a conserved tyrosine residue (Tyr323 in\n                    <jats:italic>Rp<\/jats:italic>\n                    NiR) located in the tethering linker between the fused domain and the core enzyme. To gain insight into this control, we have undertaken targeted mutations of\n                    <jats:italic>Rp<\/jats:italic>\n                    NiR to probe the so-called primary proton channel and perturb putative electron transfer routes from the haem to the `gatekeeper' Tyr323 and to the T1Cu centre. The resolution of our crystallographic data to better than 1.2\u2005\u00c5 enabled us to apply unrestrained\n                    <jats:italic>SHELXL<\/jats:italic>\n                    refinement of the structures. Our data provide a significant advance in our understanding of catalysis and modulation of electron transfer in these tethered systems, with wider implications for these fundamental processes in other protein complexes.\n                  <\/jats:p>","DOI":"10.1107\/s2052252526004549","type":"journal-article","created":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T07:59:56Z","timestamp":1780646396000},"page":"498-513","update-policy":"https:\/\/doi.org\/10.1107\/cm_01","source":"Crossref","is-referenced-by-count":0,"title":["Functional role of a tethered domain as a naturally fused cognate partner is demonstrated in a three-domain copper nitrite reductase"],"prefix":"10.1107","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6218-5368","authenticated-orcid":true,"given":"Nopphon","family":"Petchyam","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04xs57h96","id-type":"ROR","asserted-by":"publisher"}],"department":["Molecular Biophysics Group, Life Sciences Building, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allegra","family":"Mbouku","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04xs57h96","id-type":"ROR","asserted-by":"publisher"}],"department":["Molecular Biophysics Group, Life Sciences Building, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7248-6789","authenticated-orcid":true,"given":"Robert R.","family":"Eady","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04xs57h96","id-type":"ROR","asserted-by":"publisher"}],"department":["Molecular Biophysics Group, Life Sciences Building, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2854-4718","authenticated-orcid":true,"given":"S. Samar","family":"Hasnain","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04xs57h96","id-type":"ROR","asserted-by":"publisher"}],"department":["Molecular Biophysics Group, Life Sciences Building, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2779-9946","authenticated-orcid":true,"given":"Svetlana V.","family":"Antonyuk","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04xs57h96","id-type":"ROR","asserted-by":"publisher"}],"department":["Molecular Biophysics Group, Life Sciences Building, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"329","published-online":{"date-parts":[[2026,6,15]]},"reference":[{"key":"lz5078_bb1","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1042\/bj3240511","volume":"324","author":"Abraham","year":"1997","journal-title":"Biochem. 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