Electrostatic analysis of the interaction of cytochrome c with native and dimethyl ester heme-substituted cytochrome b5

Abstract

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)

NMR study of the interaction between cytochrome b5 and cytochrome c

“…These (iii) Cytochrome c and [Cr(en)313' may bind to different regions of the cytochrome bs surface. Cytochrome b5 is a highly anionic protein (net charge -9 at pH 7), and its cation-binding surface is large enough [9] to bind both cytochrome c and [Cr(en)313+ simultaneously.…”

“…That model proposed that the haem edge of cytochrome b5 was buried within the cytochrome b5-c complex with haem propionate 7 forming a salt bridge with Lys 79 of cytochrome c. Recently, measurements of the stability of complex formation between DME-cytochrome bs and cytochrome c combined with electrostatics calculations have led to the conclusion [9] that blocking of the cytochrome bs haem propionate groups (as methyl esters) can result in an alternative docking geometry for complex formation that is electrostatically isoenergetic with the Salemme complex at pH 7. For the native proteins, parallel peculations verified that the Salemme complex is the electrostatically favoured arrangement at neutrality.…”

“…Recent studies of the role of a cytochrome bs haem propionate group in complex formation with cytochrome c have led to the proposal that at least one alternative docking orientation possesses electrostatic stability equal to that of the Salemme complex under specific solution conditions [9]. The present paper reports the 'H NMR spectroscopy examination of the cytochrome bs-c interaction using the paramagnetic-reagent-competition approach described in [8].…”

A further investigation of the cytochrome b 5–cytochrome c complex

“…All these studies clearly point out that the interaction is mainly driven by electrostatic terms but open questions on the details of the interaction still remain. It has been suggested by several researchers that alternative complex geometries to the one designed by Salemme [1] should be invoked to explain experimental results, as well as the fact that more than one complex configuration could be present in solution [7,8]. Flexibility of electron transfer protein-protein complexes, as opposed to a tight specific binding, as well as local mobility have also been proposed [8,9,10].…”

“…It has been suggested by several researchers that alternative complex geometries to the one designed by Salemme [1] should be invoked to explain experimental results, as well as the fact that more than one complex configuration could be present in solution [7,8]. Flexibility of electron transfer protein-protein complexes, as opposed to a tight specific binding, as well as local mobility have also been proposed [8,9,10]. In this field, 1 H NMR spectroscopy provided evidence of the interaction through chemical shift analysis, first through hyperfine-shifted signals [11,12] and then through 2D 1 H NMR spectroscopy.…”