Perturbations at the high spin heme b center in the membrane-bound nitric oxide reductase

Abstract The effects of lowering pH from 7 to 5 on the absorption, circular dichroism (MCD) and EPR spectra were studied for Paracoccus halodenitrificans nitric oxide reductase (NOR). Intensities of the characteristic bands for the high spin heme b , that at 592 nm in the absorption spectrum and those at 591 (+) and 606 (−) in the MCD spectrum decreased considerably. Concomitant cryogenic EPR spectrum indicated a drastic increase in the signal intensity due to the high spin heme b at g ∼6, of which less than 5% had been EPR detectable at pH 7. Cyanide (×40) bound to the high spin heme b center in the reduced NOR irrespective of pH, while a much larger amount of azide (×1000) was necessary to bind to the reduced NOR at an acidic pH, ca. 5. Based on these results the structure and function of the high spin heme b center as the active site of NOR was discussed.