Mapping the Electron Transfer Interface between Cytochrome b₅ and Cytochrome c

Abstract

To characterize the cytochrome b(5) (Cyt b(5))-cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b(5) have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b(5)-Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b(5) have been removed. The results support a dynamic docking paradigm for Cyt b(5)-Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b(5) and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b(5)-Cyt c system and the Cyt b(5)-Zn-Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…This conclusion may hold in ViVo and suggests that electron transport in real biological systems may be faster in situations where the components of a transient complex are not connected together in a single, well defined manner. [347][348][349][350][351][352][353][354][355][356] Hildebrandt and co-workers have also emphasized the similarity between self-assembled monolayers and biological membranes. 357 In the case of bare metal electrodes, the protein-electrode interaction is not expected to mimic the physiological situation.…”

“…This may be reminiscent of a common situation for inter-protein electron transfer. Indeed, it is now admitted that distributions of orientations and electron transfer rates also exist in protein-protein electron transfers, as evidenced in NMR experiments, [347][348][349][350][351][352][353][354] soft-docking, 355 and molecular dynamics simulations, 356 but the heterogeneity of intermolecular ET rate constants is likely to escape detection in traditional kinetic measurements. In contrast, the distribution of interfacial ET rate constants is evident from the shape of the PFV data, and this illustrates an advantage of measuring the complete dependence of rate on driving force rather than a single value of the rate as in homogeneous kinetics.…”

Characterization and Calculation of a Cytochrome c−Cytochrome b5 Complex Using NMR Data

“…While Lys72 of cyt c can make salt bridges with both Glu56 and Asp60 of cyt b 5 The molecular interface of the three solutions in cluster A is consistent with the earlier observation of the importance of charged residues 43, 44, 48, 56, and 60 of cyt b 5 and the heme 6-propionate in the complex formation. Replacement of charged residues 43, 44, 48, and 60 of cyt b 5 with their neutral analogues (8) and of residues 44, 48, 54, and 60 (as well as multiple replacements) with Ala by site-directed mutagenesis results in an increase in the dissociation constant of the complex (12,57). However, removal of Glu44, Glu48, and Asp60 and modification of the heme propionate yield an only 14% decrease in the free energy of association with cyt c (9).…”

Electron Transfer Interactome of Cytochrome c

“…However, crystal structures of ET protein complexes show very few charge-charge or polar contacts across the interfaces, with most of the interactions dominated by van der Waals (vdW) forces [13]. Moreover, in several systems, mutagenesis of the charged residues expected to stabilize transient protein-protein complexes shows little or no effect on the binding affinity and the ET rate [14]–[16]. Thus, contrary to the conclusions of early modeling studies [5], [6], it appears that ET complexes are not optimized for intermolecular electrostatic interactions, which is thought to facilitate rapid dissociation required for a high turnover [13].…”