Reduction of ferrylmyoglobin by the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) in aqueous solution and during freezing

The hypervalent muscle pigment ferrylmyoglobin, formed by activation of met myoglobin by hydrogen peroxide, was found to be reduced in a second-order r eaction by N-tert-butyl-alpha-phenylnitrone (PBN, often used as a spin trap ). In acidic aqueous solution at ambient temperature, the reduction is rela tively slow (Delta H-double dagger = 65+/-2k KJ . mol(-1) and Delta S-doubl e dagger = -54+/-7 J . mol(-1) . K-1 for pH = 5.6), but phase transitions d uring freezing of the buffered solutions accelerates the reaction between f errylmyoglobin and PEN. Ln these heterogenous systems at low temperature (b ut not when ice-formation was inhibited by glycerol), a PEN-derived radical intermediate was detected by ESR-spectroscopy, identified as a nitroxyl ra dical by a parallel nitrogen hyperfine coupling constant of 31.8 G, and fro m microwave power saturation behavior concluded not to be located in the he me-cleft of the protein. The acceleration of the reaction is most likely ca used by a lowering of the pH during the freezing of the buffered solutions whereby ferrylmyoglobin becomes more oxidizing.