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Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) does not disproportionate hydroxylamine to ammonia and nitrite, despite a strongly favorable driving force. Biochemistry 2014 Apr 08;53(13):2136-44

Date

03/22/2014

Pubmed ID

24645742

Pubmed Central ID

PMC5047060

DOI

10.1021/bi401705d

Scopus ID

2-s2.0-84898037971 (requires institutional sign-in at Scopus site)   11 Citations

Abstract

Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV-visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2(+) moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

Author List

Youngblut M, Pauly DJ, Stein N, Walters D, Conrad JA, Moran GR, Bennett B, Pacheco AA

Authors

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University
Arsenio Andrew Pacheco in the Chemistry and Biochemistry department at University of Wisconsin - Milwaukee




MESH terms used to index this publication - Major topics in bold

Ammonia
Catalytic Domain
Cytochromes a1
Cytochromes c1
Hydroxylamine
Nitrate Reductases
Nitrites
Shewanella
Thermodynamics