Magnetic circular dichroism studies of hemoglobin. The reduction of ferrihemoglobin by ferrocytochrome b5 and characterization of the high-spin hydroxy species of mixed-valence hemoglobin.
The final step in the erythrocyte methemoglobin reduction pathway, the transfer of an electron from cytochrome b5 to methemoglobin, has been studied using magnetic circular dichroism spectroscopy. Spectral analysis allowed us to determine accurately the concentration of each redox species in mixtures of the two heme-proteins and to follow simultaneously the kinetics of the appearance or disappearance of each of these species during reduction reactions. Our analysis detected a substantial increase in the high-spin hydroxymethemoglobin species in the partially reduced bovine hemoglobin tetramer. This species was sensitive to the degree of reduction and pH, and was spectrally similar to fluoride methemoglobin. At pH 7.8, 100% of the hydroxide component of methemoglobin was in the high-spin form when two or more subunits were in the ferrous form. Kinetic analysis of bovine methemoglobin reduction yielded values for the apparent first-order rates for the tetrameric species possessing four, three, two, and one ferric subunit. Further analysis showed that the reduction kinetics can also be described by an equilibrium state, pure competitive inhibition model for enzyme catalysis in which ferrous and ferric subunits of hemoglobin compete for cytochrome b5. This analysis generated a KD that depends on ionic strength and hemoglobin tetramer conformation, a Vmax that was independent of these factors, and an inhibition constant that was equal to KD. This model is consistent with the hypothesis that the reduction of methemoglobin can be separated into two steps, the ionic interaction between cytochrome b5 and hemoglobin and the electron transfer.
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D006454:Hemoglobins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008280:Magnetics; D008433:Mathematics; D008706:Methemoglobin / Q000378:metabolism*
D003573:Cytochrome b Group; D006454:Hemoglobins; D008706:Methemoglobin; D015786:Cytochromes b5