Abstract
An active fraction of hydroxylamine-cytochrome c reductase (NH_2OH-cyt. c reductase) was isolated from N. europaea. The production of nitrite from hydroxylamine by this fraction was subsequently evaluated. The fraction with NH_2OH-cyt. c reductase and nitrite synthetase activities was obtained using DEAE-sepharose column chromatography. The fraction, f13, contains cytochrome and related compounds with a NH_2OH-cyt. c reductase activity. The activity from this fraction was higher than that of fraction F. 6 obtained by DEAE-cellulose column chromatography. The purity of fraction f13 was higher than that of F. 6, and the same enzyme protein was detected in both fractions. The cytochrome in fraction f13 was identified haem c. using the absorption spectrum of pyridine ferrohaemochrome and extracted haem. The f13 fraction was subsequenty divided into three protein fractions by gel filtration over a sephadex G-100 column, affording apl fraction exhibiting activities of both enzymes. Thispl fraction was further divided into five protein fractions using polyacrylamide gel electrophoresis. Activity representative of both enzymes was found in the a3 fraction. SDS-polyacrylamide gel electrophoresis revealed that a3 fraction consists of two subunits with a molecular weight of 175,000. Several studies were carried out on this a3 fraction. The oxidation reaction of hydroxlamine revealed the additive effect of cytochrome c and cytochrome c oxidase. A correlation was noted between the quantity of oxidized hydroxylamine and the quantity of nitrite produced. Even in the presence of excess cytochrome c, the absence of cytochrome c oxidase was related to a 50% reduction in the amount of nitrite formed from hydroxylamine. The reaction ratio of reduced cytochrome c to hydroxylamine was 1:3.
