Nitrosylation of c heme in cd(1)-nitrite reductase is enhanced during catalysis

The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d(1) heme per mono-mer (cd(1)NiR), encoded by the nirS gene. For many years, the evidence of a link between NO and this hemeprotein represented a paradox, given that NO was known to tightly bind and, possibly, inhibit hemeproteins, including cd(1)NiRs. It is now established that, during catalysis, cd(1)NiRs diverge from "canonical" hemeproteins, since the product NO rapidly dissociates from the ferrous d(1) heme, which, in turn, displays a peculiar "low" affinity for NO (K-D = 0.11 microM at pH 7.0). It has been also previously shown that the c heme reacts with NO at acidic pH but c heme nitrosylation was not extensively investigated, given that in cd(1)NiR it was considered a side reaction, rather than a genuine process controlling catalysis. The spectroscopic study of the reaction of cd(1)NiR and its semi-apo derivative (containing the sole c heme) with NO reported here shows that c heme nitrosylation is enhanced during catalysis; this evidence has been discussed in order to assess the potential of c heme nitrosylation as a regulatory process, as observed for cytochrome c nitrosylation in mammalian mitochondria.