On the purification of nitrite reductase from [formula omitted] and its reaction with nitrite under reducing conditions

Abstract

Nitrite reductase (cytochrome cd ) from T. denitrificans has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from P. aeruginosa . EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both c - and d -hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a 14N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant. To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.