Time-resolved infrared spectroscopy reveals a stable ferric heme-NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase with nitrite

Cytochrome cd(1) is a respiratory enzyme that catalyzes the physiological o ne-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, ea ch monomer containing one c-type cytochrome center and one active site d(1) heme, We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d(1)-NO complex (formally d(1)Fe(II)-NO+) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome ed, and nitrite, in the absence of excess reductant, The Fe-(NO )-N-14 nu>(*) over bar * (NO) stretching mode is observed at 1913 cm(-1) wi th the corresponding Fe-(NO)-N-15 band at 1876 cm(-1). This d(1) heme-NO co mplex is still readily observed after 15 min. EPR and visible absorption sp ectroscopic data show that within 4 ms of the initiation of the reaction, n itrite is reduced at the d(1) heme, and a cFe(III) d(1)Fe(II)-NO complex is formed. Over the next 100 ms there is an electron redistribution within th e enzyme to give a mixed species, 55% cFe(III) d(1)Fe(II)-NO and 45% cFe(II ) d(1)Fe(II)-NO+. No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product rel ease by the enzyme. Implications for the mechanism of P. pantotrophus cytoc hrome cd(1) are discussed.