Nitrite dismutase reaction mechanism: kinetic and spectroscopic investigation of the interaction between nitrophorin and nitrite.
J Am Chem Soc
; 137(12): 4141-50, 2015 Apr 01.
Article
en En
| MEDLINE
| ID: mdl-25751738
ABSTRACT
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 73 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.
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MEDLINE
Asunto principal:
Rhodnius
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Proteínas y Péptidos Salivales
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Proteínas de Insectos
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Hemoproteínas
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Nitritos
Idioma:
En
Revista:
J Am Chem Soc
Año:
2015
Tipo del documento:
Article