Time-resolved infrared spectroscopy reveals a stable ferric heme-NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with nitrite

Abstract

Cytochrome cd1 is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d1 heme. We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d1-NO complex (formally d1Fe(II)-NO+) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome cd1 and nitrite, in the absence of excess reductant. The Fe-14NO v(NO) stretching mode is observed at 1913 cm-1 with the corresponding Fe-15NO band at 1876 cm-1. This d1 heme-NO complex is still readily observed after 15 min. EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d1 heme, and a cFe(III) d1Fe(II)-NO complex is formed. Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III) d1Fe(II)-NO and 45% cFe(II) d1Fe(II)-NO+. No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme. Implications for the mechanism of P. pantotrophus cytochrome cd1 are discussed.