{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T01:27:34Z","timestamp":1786584454257,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,10]]},"group-title":"Biochemistry","reference-count":83,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas, AR","award":["PIP 706"],"award-info":[{"award-number":["PIP 706"]}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["GM30228"],"award-info":[{"award-number":["GM30228"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Nitrite can be reduced to nitric oxide (NO) by several heme- and molybdenum-containing proteins, including mitochondrial cytochrome c oxidase (Cco). This activity, designated Cco\/NO, has been implicated in hypoxic signaling, but its regulation and quantitative significance relative to other NO-producing systems remain uncertain. We examined its modulation by adenine nucleotides using detergent-solubilized yeast and mouse brain mitochondria supplied with 1 mM nitrite and an ascorbate\/TMPD\/cytochrome c electron-donor system. ADP and ATP differentially modulated Cco\/NO activity, and ADP extended measurable NO formation across the entire oxygen range tested, up to the assay ceiling of 175 \u00b5M O\u2082. Nucleotide regulation was also isoform-dependent: ATP slightly inhibited Va-containing Cco but strongly stimulated Vb-containing Cco under anoxic conditions. Rates normalized to cytochrome aa\u2083 demonstrate multi-turnover nitrite-reductase capacity under these substrate-driven assay conditions. Both the cellular ADP\/ATP ratio and subsequently assayed Cco\/NO activity increased transiently following a hypoxic shift. These findings establish metabolic and isoform-dependent gating of the catalytic capacity of Cco\/NO; they do not establish its fractional contribution to total cellular NO or its operation at physiological nitrite concentrations in intact, coupled mitochondria.<\/jats:p>\n                <jats:p>This research was supported by CONICET Grant PIP 706 (research team member P.R.C.) and National Institutes of Health Grant GM30228 to R.O.P.<\/jats:p>","DOI":"10.64898\/2026.08.09.743791","type":"posted-content","created":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T08:45:17Z","timestamp":1786437917000},"source":"Crossref","is-referenced-by-count":0,"title":["Mitochondrial Signaling: Nitric Oxide Synthesis by Cytochrome c Oxidase and Its Oxygen Sensitivity Are Modulated by Adenine Nucleotides"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2983-9294","authenticated-orcid":false,"given":"Pablo R.","family":"Castello","sequence":"first","affiliation":[{"name":"Facultad de Ciencias Exactas y Naturales, Universidad de Belgrano, and CONICET, Buenos Aires, Argentina. Zabala 1324, 1428 Ciudad de Buenos Aires, Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kerri A.","family":"Ball","sequence":"additional","affiliation":[{"name":"Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert O.","family":"Poyton","sequence":"additional","affiliation":[{"name":"Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026081217503720000_2026.08.09.743791v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2022.06.010"},{"key":"2026081217503720000_2026.08.09.743791v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/S0891-5849(02)00892-4"},{"key":"2026081217503720000_2026.08.09.743791v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro1004"},{"key":"2026081217503720000_2026.08.09.743791v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(04)00131-X"},{"key":"2026081217503720000_2026.08.09.743791v1.5","doi-asserted-by":"publisher","DOI":"10.1126\/science.1108228"},{"key":"2026081217503720000_2026.08.09.743791v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/ncb1268"},{"key":"2026081217503720000_2026.08.09.743791v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/ncb0705-645"},{"key":"2026081217503720000_2026.08.09.743791v1.8","doi-asserted-by":"publisher","DOI":"10.1152\/ajpheart.00374.2008"},{"key":"2026081217503720000_2026.08.09.743791v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/nm0895-804"},{"key":"2026081217503720000_2026.08.09.743791v1.10","doi-asserted-by":"publisher","DOI":"10.1002\/med.20151"},{"key":"2026081217503720000_2026.08.09.743791v1.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.freeradbiomed.2011.05.037"},{"key":"2026081217503720000_2026.08.09.743791v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.niox.2009.12.004"},{"key":"2026081217503720000_2026.08.09.743791v1.13","doi-asserted-by":"publisher","DOI":"10.1038\/nrd2466"},{"key":"2026081217503720000_2026.08.09.743791v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2006.02.011"},{"key":"2026081217503720000_2026.08.09.743791v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2012.03.050"},{"key":"2026081217503720000_2026.08.09.743791v1.16","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0709461105"},{"key":"2026081217503720000_2026.08.09.743791v1.17","first-page":"154","article-title":"Therapeutic photobiomodulation: nitric oxide and a novel function of mitochondrial cytochrome c oxidase","volume":"11","year":"2011","journal-title":"Discov. 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