D1 haem biogenesis - Assessing the roles of three nir gene products

The synthesis of the modified tetrapyrrole known as d1 haem requires several dedicated proteins which are coded for by a set of genes that are often found adjacent to the structural gene, nirS, for cytochrome cd 1 nitrite reductase. NirE, the product of the first gene in the nir biogenesis operon, was anticipated to catalyse the conversion of uroporphyrinogen III into precorrin-2; this was confirmed, but it was shown that this enzyme is less sensitive to product inhibition than similar enzymes that function in other biosynthetic pathways. Sequence analysis suggesting that one of these proteins, NirN, is a c-type cytochrome, and has similarity to the part of cytochrome cd1 that binds d1, was validated by recombinant production and characterization of NirN. A NirN-d1 haem complex was demonstrated to release the cofactor to a semi-apo form of cytochrome cd1 from which d1 was extracted, suggesting a role for NirN in the assembly of cytochrome cd1 (NirS). However, inactivation of nirN surprisingly led to only a marginal attenuation of growth of Paracoccus pantotrophus under anaerobic denitrifying conditions. As predicted, NirC is a c-type cytochrome; it was shown in vitro to be an electron donor to the NirN-d1 complex