The stability of the complex formed between cytochrome c and dimethyl ester heme substituted cytochrome b5 (DME-cytochrome b5) has been determined under a variety of experimental conditions to evaluate the role of the cytochrome b5 heme propionate groups in the interaction of the two native proteins. Interaction between cytochrome c and the modified cytochrome b5 was found to produce a difference spectrum in the visible range that is very similar to that generated by the interaction of the native proteins and that can be used to monitor complex formation between the two proteins. At pH 8 [25 degrees C (HEPPS), I = 5 mM], DME-cytochrome b5 and cytochrome c form a 1:1 complex with an association constant KA of 3 (1) X 10(6) M-1. This pH is the optimal pH for complex formation between these two proteins and is significantly higher than that observed for the interaction between the two native proteins. The stability of the complex formed between DME-cytochrome b5 and cytochrome c is strongly dependent on ionic strength with KA ranging from 2.4 X 10(7) M-1 at I = 1 mM to 8.2 X 10(4) M-1 at I = 13 mM [pH 8.0 (HEPPS), 25 degrees C]. Calculations for the native, trypsin-solubilized form of cytochrome b5 and cytochrome c confirm that the intermolecular complex proposed by Salemme [Salemme, F. R. (1976) J. Mol. Biol. 102, 563] describes the protein-protein orientation that is electrostatically favored at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)
The interaction between cytochrome c and the tryptic fragment of cytochrome b5 has been found to produce a difference spectrum in the Soret region with a maximum absorbance at 416 nm. The intensity of this difference has been used to determine the stoichiometry of complex formation and the stability of the complex formed. At pH 7.0 [25 degrees C (phosphate), mu = 0.01 M], the two proteins were found to form a 1:1 complex with an association constant, KA, of 8(3) x 10(4) M-1. The stability of the complex was found to be strongly dependent on ionic strength with KA increasing to 4(3) x 10(6) M-1 at mu = 0.001 M [25 degrees C, pH 7.0 (phosphate)]. Analysis of the dependence of KA on pH from pH 6.5 to 8 demonstrated that this complex is maximally stable between pH 7 and 8 or about midway between the isoelectric points of the two proteins. Analysis of the temperature dependence of KA revealed that formation of the complex between the two proteins is largely entropic in origin with delta Ho = 1 +/- 3 kcal/mol and delta So = 33 +/- 11 eu [pH 7.0 (phosphate), mu = 0.001 M]. This result may be explained either by the model of Clothia and Janin [Clothia, C., & Janin, J. (1975) Nature (London) 256, 705] in terms of extensive solvent reorganization upon complexation or by the model of Ross and Subramanian [Ross, P. D., & Subramanian, S. (1981) Biochemistry 20, 3096] in which the negative enthalpic and entropic contributions of short-range protein-protein interactions are offset by proton release.
The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990). Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization. Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein. The midpoint reduction potential of this protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with delta H degrees = -3.2 +/- 0.1 kcal/mol and delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10). The 1D 1H NMR spectrum of the erythrocytic ferricytochrome indicates that 90% of the protein binds heme in the "major" orientation and 10% of the protein binds heme in the "minor" orientation (pH 7.0, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium.
Two potentiometric methods have been used to study the pH-dependent changes in proton binding that accompany complex formation between cytochrome c and cytochrome b5. With one method, the number of protons bound or released upon addition of one cytochrome to the other has been measured as a function of pH. The results from these studies are correlated with the complexation-induced difference titration curve calculated from the titration curves of the preformed complex and of the individual proteins. Both methods demonstrate that complex formation at acid pH is accompanied by proton release, that complex formation at basic pH is accompanied by proton uptake, and that the change in proton binding at neutral pH, where stability of complex formation is maximal, is relatively small. Under all conditions studied, the stoichiometry of cytochrome c-cytochrome b5 complex formation is 1:1 with no evidence of higher order complex formation. Although the dependence of complex formation on pH for interaction between different species of cytochrome c and cytochrome b5 are qualitatively similar, they are quantitatively different. In particular, complex formation between yeast iso-1-cytochrome c and lipase-solubilized bovine cytochrome b5 occurs with a stability constant that is 10-fold greater than observed for the other two pairs of proteins under all conditions studied. Interaction between these two proteins is also significantly less dependent on ionic strength than observed for complexes formed by horse heart cytochrome c with either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)
Rate constants for electron transfer in the complex between recombinant rat mitochondrial outer membrane cytochrome b5 or the tryptic fragment of bovine liver cytochrome b5 and horse mitochondrial cytochrome c were measured by laser flash photolysis of 5-deazariboflavin-EDTA solutions. When an excess of cytochrome b5 was titrated with increasing amounts of cytochrome c at low ionic strength and electron transfer was initiated by a laser flash, both proteins were rapidly reduced by deazariboflavin semiquinone. The initial photoreduction was followed by a slower second-order reduction of b5 complexed oxidized cytochrome c by free reduced cytochrome b5. At an 8:1 ratio of cytochromes b5 to c, the pseudo-first-order rate constant for reduction of complexed cytochrome c increased 3-5-fold between ionic strengths of 5 and 40 mM, and then dropped precipitously at higher ionic strengths. The ionic strength dependent increase in rate constant is likely to be due to relief of steric hindrance via rearrangement of cytochrome c in the complex. The reaction rate showed no sign of saturation at any ionic strength, indicating a first-order rate constant greater than 10(4) s-1 within a transient ternary protein complex; i.e., interprotein electron transfer approaches the largest values previously reported for the stable binary protein complex (approximately 4 x 10(5) s-1). Our results emphasize the flexibility of electron-transfer protein complexes, which had previously been modeled in a single conformation with specific salt bridges. It appears that a variety of orientations can exist within such protein-protein complexes and that the population of conformations changes with ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.