Interaction between cytochrome b5 and human methemoglobin.
Complex formation between purified human methemoglobin and the tryptic fragment of bovine liver cytochrome b5 has been demonstrated by the observation of a difference spectrum produced on mixing the two proteins. The intensity of this difference spectrum has been used to determine the stoichiometry of the complex formed and its stability under a variety of conditions. At pH 6.2 [25 degrees C (cacodylate buffer), mu = 2 mM], the complex has a stoichiometry of 1:1 (heme: heme) with a stability constant, KA, of (3 +/- 2) X 10(5) M(-1). This stability constant is dependent on ionic strength, decreasing to a value of (9 +/- 3) X 10(3) M(-1) at mu = 12 mM [pH 6.2 (cacodylate buffer), 25 degrees C]. Analysis of this dependence by fitting the data to a form of the Debye-Hückel 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 two proteins. Determination of the pH dependence of KA revealed that the complex is most stable at slightly acidic pH (pH 6.0-6.2) or, in other words, at a pH that is approximately midway between the isoelectric 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 [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 one cytochrome b5 by means of complementary charge interactions between oppositely charged groups on the two proteins. The probable sites of cytochrome b5 binding on hemoglobin are discussed.
D000818:Animals; D002417:Cattle; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008706:Methemoglobin / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D013053:Spectrophotometry; D014357:Trypsin
D003573:Cytochrome b Group; D010446:Peptide Fragments; D008706:Methemoglobin; D015786:Cytochromes b5; D014357:Trypsin