Nitrite Dismutase Reaction Mechanism: Kinetic and Spectroscopic Investigation of the Interaction between Nitrophorin and Nitrite

• Published in: Journal of the American Chemical Society 2015-04, Vol.137 (12), p.4141-4150
• Main Authors: He, Chunmao, Howes, Barry D, Smulevich, Giulietta, Rumpel, Sigrun, Reijerse, Edward J, Lubitz, Wolfgang, Cox, Nicholas, Knipp, Markus
• Format: Article
• Language: English
• Subjects: Animals; Electron Spin Resonance Spectroscopy; Hemeproteins - chemistry; Hemeproteins - metabolism; Insect Proteins - chemistry; Insect Proteins - metabolism; Iron Compounds - chemistry; Iron Compounds - metabolism; Kinetics; Models, Molecular; Nitrites - chemistry; Nitrites - metabolism; Nitrogen Oxides - chemistry; Nitrogen Oxides - metabolism; Rhodnius - chemistry; Rhodnius - metabolism; Salivary Proteins and Peptides - chemistry; Salivary Proteins and Peptides - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja512938u

Nitrite is an important metabolite in the physiological pathways of NO and other nitrogen oxides in both enzymatic and nonenzymatic reactions. The ferric heme b protein nitrophorin 4 (NP4) is capable of catalyzing nitrite disproportionation at neutral pH, producing NO. Here we attempt to resolve its disproportionation mechanism. Isothermal titration calorimetry of a gallium­(III) derivative of NP4 demonstrates that the heme iron coordinates the first substrate nitrite. Contrary to previous low-temperature EPR measurements, which assigned the NP4-nitrite complex electronic configuration solely to a low-spin (S = 1/2) species, electronic absorption and resonance Raman spectroscopy presented here demonstrate that the NP4-NO2 – cofactor exists in a high-spin/low-spin equilibrium of 7:3 which is in fast exchange in solution. Spin-state interchange is taken as evidence for dynamic NO2 – coordination, with the high-spin configuration (S = 5/2) representing the reactive species. Subsequent kinetic measurements reveal that the dismutation reaction proceeds in two discrete steps and identify an {FeNO}7 intermediate species. The first reaction step, generating the {FeNO}7 intermediate, represents an oxygen atom transfer from the iron bound nitrite to a second nitrite molecule in the protein pocket. In the second step this intermediate reduces a third nitrite substrate yielding two NO molecules. A nearby aspartic acid residue side-chain transiently stores protons required for the reaction, which is crucial for NPs’ function as nitrite dismutase.