Abstract
Heme d1 plays an important role in denitrification as the essential cofactor of the cytochrome cd1 nitrite reductase NirS. At present, the biosynthesis of heme d1 is only partially understood. The last step of heme d1 biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d1. Instead, the NirS purified from this strain contains the heme d1 precursor dihydro-heme d1 lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d1 was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d1 and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d1.