Purification and characterization of the single-component nitric oxide reductase from Ralstonia eutropha H16

• Published in: FEBS letters 1999-10, Vol.460 (1), p.6-10
• Main Authors: Cramm, Rainer, Pohlmann, Anne, Friedrich, Bärbel
• Format: Article
• Language: English
• Subjects: Ascorbic Acid - chemistry; Bacterial Proteins - chemistry; Cupriavidus necator - enzymology; Denitrification; Electron Spin Resonance Spectroscopy; Electron Transport; Electrophoresis, Polyacrylamide Gel; EPR, electron paramagnetic resonance; Heme - chemistry; Heme-copper oxidase; Hemeproteins - chemistry; Iron - chemistry; Kinetics; Methylphenazonium Methosulfate - chemistry; Nitric oxide reductase; NTA, nitrilotriacetic acid; Oxidoreductases - chemistry; PMS, phenazine methosulfate; Quinol oxidase; Ralstonia eutropha; Spectrophotometry; Tetramethylphenylenediamine - chemistry; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine; Vitamin K - chemistry
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(99)01315-0

Nitric oxide (NO) reductase was purified from Ralstonia eutropha (formerly Alcaligenes eutrophus) using a two step chromatographic procedure. Unlike the common NO reductases, the enzyme consists of a single subunit of 75 kDa which contains both high-spin and low-spin heme b, but lacks heme c. One additional iron atom, probably a ferric non-heme iron, was identified per enzyme molecule. Whereas reduced cytochrome c was ineffective as electron donor, NO was reduced at a specific activity of 2.3 μmol/min per mg of protein in the presence of 2-methyl-1,4-naphthoquinol.