Isolation of Paracoccus denitrificans cytochrome cd1: Comparative kinetics with other nitrite reductases

Abstract

The dissimilatory nitrite reductase of the cytochrome cd 1 type was purified from Paracoccus denitrificans (ATCC 13543) by a novel procedure that avoided conventional ion-exchange techniques. The characterization of this enzyme was extended to include amino acid composition, extinction coefficients, and kinetic properties not previously reported. Cytochromes cd 1 from Alicaligenes faecalis and Pseudomonas aeruginosa were also isolated and assayed with electron donor proteins. The enzymes from all three sources were shown to obey the same integrated rate law. Cross-reactivities were measured in which a reduced donor protein from one strain was assayed with cytochrome cd 1 from another strain using nitrite as ultimate acceptor. Donors included c-type cytochromes and azurins. In general, the enzymes showed specificity for a donor from the same strain; interspecies cross-reactions were typically slower on the order of 10-fold than corresponding native rates. Notable exceptions were Paracoccus cytochrome cd 1, which alone reacted with eukaryotic horse cytochrome c at appreciable rates, and the Pseudomonas cd 1-Alcaligenes c554 reaction, which was 4-fold faster than the native Alcaligenes cd 1-Alcaligenes c554 reaction. For all three enzymes, competitive kinetics were measured in which the alternative substrates, nitrite and oxygen, competed for enzyme in the same assay. It was found that the competitive kinetics were dominated by nonenzymatic reactions involving an enzyme product, nitric oxide.