Interaction between cytochrome b5 and human methemoglobin

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Abstract
Complex formation between purified human methemoglobin and the tryptic fragment of bovine liver cytochrome b5 was demonstrated by the observation of a difference spectrum produced on mixing the 2 proteins. The intensity of this difference spectrum was used to determine the stoichiometry of the complex formed and its stability under a variety of conditions. At pH 6.2 [25.degree. C (cacodylate buffer), .mu. = 2 mM], the complex has a stoichiometry of 1:1 (heme: heme) with a stability constant KA, of (3 .+-. 2) .times. 105 M-1. This stability constant is dependent on ionic strength, decreasing to a value of (9 .+-. 3) .times. 103 M-1 at .mu. = 12 mM [pH 6.2 (cacodylate buffer), 25.degree. C]. Analysis of this dependence by fitting the data to a form of the Debye-Hueckel equation produces a charge product of -64 .+-. 14, which is in reasonable agreement with the value anticipated on the basis of the amino acid sequences of the 2 proteins. Determination of the pH dependence of KA revealed that the complex is most stable at slightly acidic pH (pH 6.0-6.2), i.e., at a pH that is approximately midway between the isoeleectric pH values of cytochrome b5 and methemoglobin. The variation of KA with temperature is consistent with .DELTA.H.degree. = -10 .+-. 3 kcal/mol and .DELTA.S.degree. = -12 .+-. 10 eu [entropy units] [pH 6.2 (cacodylate buffer), .mu. = 5 mM]. Together, these results generate a model for cytochrome b5-methemoglobin interaction in which each hemoglobin subunit binds 1 cytochrome b5 by means of complementary charge interactions between oppositely charged groups on the 2 proteins. The probable sites of cytochrome b5 binding on Hb are discussed.