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 metmyoglobin by hydrogen peroxide, was found to be reduced in a second-order reaction by N-tert-butyl-alpha-phenylnitrone (PBN, often used as a spin trap). In acidic aqueous solution at ambient temperature, the reduction is relatively slow (deltaH++ = 65+/-2kJ x mol(-1) and deltaS++ = -54+/-7 J x mol(-1). K(-1) for pH = 5.6), but phase transitions during freezing of the buffered solutions accelerates the reaction between ferrylmyoglobin and PBN. In these heterogenous systems at low temperature (but not when ice-formation was inhibited by glycerol), a PBN-derived radical intermediate was detected by ESR-spectroscopy, identified as a nitroxyl radical by a parallel nitrogen hyperfine coupling constant of 31.8 G, and from microwave power saturation behavior concluded not to be located in the heme-cleft of the protein. The acceleration of the reaction is most likely caused by a lowering of the pH during the freezing of the buffered solutions whereby ferrylmyoglobin becomes more oxidizing.

Acid-catalysed autoreduction of ferrylmyoblobin in aqueous solution studied by freeze quenching and ESR spectroscopy.

Decay of the hypervalent muscle pigment ferrylmyoglobin, formed by activation of metmyoglobin by hydrogen peroxide, was found, when studied by a combination of ESR and UV/VIS spectroscopy in aqueous solution at physiological pH, to proceed by parallel second- and first-order kinetics. At pH below 6.5 a sharp ESR signal (g = 2.003) with an increasing intensity for decreasing pH were observed in solutions frozen in liquid nitrogen, and a broad signal (g = 2.005) was seen throughout the studied pH range also in frozen solutions. The g = 2.005 signal is suggested to arise from an intermediate formed in an intramolecular rate-determining electron-transfer in ferrylmyoglobin, whereas the g 2.003 signal is caused by a radical formed in a proton-assisted electron-transfer initiating the specific acid-catalysed autoreduction.

Protein binding in deactivation of ferrylmyoglobin by chlorogenate and ascorbate.

Kinetics of reduction of iron(IV) in ferrylmyoglobin by chlorogenate in neutral or moderately acidic aqueous solutions (0.16 M NaCl) to yield metmyoglobin was studied using stopped flow absorption spectroscopy. The reaction occurs by direct bimolecular electron transfer with (2.7 +/- 0.3) x 10(3) M(-)(1).s(-)(1) at 25.0 degrees C (DeltaH( )(#) = 59 +/- 6 kJ.mol(-)(1), DeltaS(#) = 15 +/- 22 J. mol(-)(1).K(-)(1)) for protonated ferrylmyoglobin (pK(a) = 4.95) and with 216 +/- 50 M(-)(1).s(-)(1) (DeltaH( )(#) = 73 +/- 8 kJ. mol(-)(1), DeltaS( )(#) = 41 +/- 30 J.mol(-)(1).K(-)(1)) for nonprotonated ferrylmyoglobin in parallel with reduction of a chlorogenate/ferrylmyoglobin complex by a second chlorogenate molecule with (8.6 +/- 1.1) x 10(2) M(-)(1).s(-)(1) (DeltaH( )(#) = 74 +/- 8 kJ.mol(-)(1), DeltaS( )(#) = 59 +/- 28 J.mol(-)(1).K(-)(1)) for protonated ferrylmyoglobin and with 61 +/- 9 M(-)(1).s(-)(1) (DeltaH( )(#) = 82 +/- 12 kJ.mol(-)(1), DeltaS( )(#) = 63 +/- 41 J. mol(-)(1).K(-)(1)) for nonprotonated ferrylmyoglobin. Previously published data on ascorbate reduction of ferrylmyoglobin are reevaluated according to a similar mechanism. For both protonated and nonprotonated ferrylmyoglobin the binding constant of chlorogenate is approximately 300 M(-)(1), and the modulation of ferrylmyoglobin as an oxidant by chlorogenate (or ascorbate) leads to a novel antioxidant interaction for reduction of ferrylmyoglobin by ascorbate in mixtures with chlorogenate.