Abstract
Nitrous oxide reductase from the denitrifying bacterium Paracoccus denitrificans has been purified very nearly in homogeneity by an anaerobic procedure
that results in a product with high specific activity. The enzyme is a dimer of about M, 144,000 composed of two subunitsof apparently equalM, and contains 4 mol of Cu per mol of subunit. The isoelectric point is 4.3; specific activity at 25 “C, pH 7.1, is 122 pmol X min” X mgof protein”; and K, is about 7 PM NzO under the sameconditions. The N20- and 02-oxid1zed formsoftheenzymehad principal absorption bandast 550 and 820 nm; the dithionite-reduced form, at 650 nm. The exinction coefficient at 550 nm for the oxidized enzyme is about 5300 (Msubunit)” X cm”. Ferricyanide-oxidized enzyme and enzyme exposed to 0 2 for a couple of days at 4 “C exhibited additional bands a t 480, 620, and 780 nm and had very low specific activities. Cu-EPR signalswere observed with oxidized and reduced forms of the enzyme with gLvalues at 2.042 and 2.055, respectively. The Oa-oxidizedenzyme had g,,and A,,values of about 2.244 and 35 gauss, respectively, based on the observation of four hyperfine lines in the g, region. The enzyme may therefore contain at least one Cu atom approximating the “Type 1” class. Spin counts againsCt u-EDTA standards suggest that 20-30% of the enzyme-bound Cu is EPR detectable in the 02-oxidizendzyme and 7-15% in the enzymeasprepared and in threeduced enzyme. Much of the Cu thus appears tobe EPR silent. Nitrousoxide reductase was observed to undergo turnover-dependent inactivation, and nitrite and fluorideamong other anions werefound to accelerate thipsrocess. In a number of characteristics, the enzyme resembles nitrous oxide reductase recently purified from Pseudomonas perfect to marina and Rhodopseudomonas sphaeroides, particularly the former. Some differences appear related to whether or not purification is carried out entirely under anaerobic conditions.