P53: Nitrite dismutase – A “nitrite-only” source for NO

Background Nitrophorins (NPs) are NO transporting proteins from the saliva of the blood-sucking insect Rhodnius prolixus . The delivery of NO is accomplished by storage on the protein’s ferriheme cofactor and the gas molecule is released when the protein is injected into a victim’s tissue. However, we demonstrated that nitrophorins can also produce NO from nitrite as the only substrate, i.e., in this reaction nitrite serves as both electron donor and electron acceptor. The enzymatic activity was termed nitrite dismutase activity and is now classified with the EC number 1.7.6.1. This study provides insight into the structural features required for the catalytic activity. Methods Proteins were recombinantly expressed in Escherichia coli cells. They were characterized by X-ray crystallography, EPR spectroscopy, UV/vis spectroscopy, resonance Raman spectroscopy, and NMR spectroscopy. The enzymatic reaction was studied by absorbance detected stopped-flow kinetics. Results The binding mode of nitrite to the NP Fe (III) is clearly different compared to that of the globins, i.e., N-bound. Contrary to the globins, the insertion of an H-donating residue (NP4(L130R)) into the protein pocket does not affect the nitrite binding mode; however, it cancels the enzymatic activity. On the other hand, mutation of the proximal Asp70 residue, which is H-bonded via a water to the iron coordinating His59, also affects the enzymatic activity, indicating that the charge distribution on the heme cofactor has a major influence on the iron reactivity. Conclusion The nitrophorins are good model systems for the characterization of the nitrite dismutase catalysis. The absence of H-donating residues and the charge density on the proximal His were identified as important structural features that are required for the nitrite dismutase reaction. Among the many heme proteins expressed in the human organism some might bear a similar catalytic activity. We suggest the group of heme peroxidases as potential candidates for enzymatic “nitrite-only” NO production. Disclosure Supported by the Deutsche Forschungsgemeinschaft (DFG), Grants KN 951-1/1 and 2.