Purification and some characteristics of nitric oxide reductase-containing vesicles from Paracoccus denitrificans

Nitric oxide reductase of Paracoccus denitrificans was purified, with the use of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) detergent, as membrane vesicles of apparent Mr = 2-3 x 10(6). Fifty percent of the protein was a peptide of Mr = 34,000. Further fractionation with sodium dodecyl sulfate (SDS) resulted in vesicles in which the peptide constituted 90-95% of the protein. This peptide, which is rich in Ala, Gly, Ser, Asx, and Glx, is considered to be the peptide of nitric oxide reductase. The CHAPSO- and SDS-fractionated preparations lost activity at 4 degrees C, pH 7.4, with half-times, respectively, of about 6 days and 4 h. Specific activities at 32 degrees C, pH 7.4, of about 0.33 mumol of NO x min-1 x mg-1 were realized after fractionation with CHAPSO in a phenazine methosulfate/ascorbate-based assay. The Km(NO) was less than or equal to 17 microM at pH 7.4. Rates decreased substantially below pH 5 and above pH 7.6. The preparations were free or almost free of cytochromes, exhibited otherwise no absorption bands in the visible region, contained no redox metals except for very small amounts of iron, were not inhibited by EDTA or some other common inhibitors of redox-metal enzymes, and were not observed to catalyze the reduction of nitrate, nitrite, or N2O. An absorption band at 274 nm in both the CHAPSO- and SDS-fractionated preparations was attributed to the presence of a solvent-soluble chromophore. N-Bromosuccinimide (NBS) inactivated the enzyme and bleached the chromophore both in the enzyme preparation and, after its purification, in 95% ethanol. NBS-inactivated enzyme could be reconstituted with purified chromophore, which alone seemed to have no nitric oxide reductase activity, but not with purified chromophore that had been reacted with NBS. Spectral changes interpretable as due to changes in redox state were not observed when enzyme was exposed to NO or certain reducing agents.