Protein-protein interactions are essential to maintain cellular function and organization and often occur by means of electrostatic interactions. These forces are determined by the electrostatic conditions within the cell, a dynamic and highly crowded environment containing ions and charged molecules.

In this thesis, the mitochondrial respiratory chain is employed as a model system to study how cellular electrostatics, specifically ionic strength, governs protein-protein interactions, with particular focus on interactions between cytochrome c and the Saccharomyces cerevisiae III₂-IV_1/2 supercomplex. The electrostatic nature of this interaction provides an ideal framework for studying ionic strength-dependent effects.

Previous data showed that at the commonly assumed “physiological” ionic strength of 150 mM monovalent salt, electron transfer within the supercomplex is mediated by 2D diffusion of a single cytochrome c molecule between complexes III₂ and IV. However, recent findings indicate that a monovalent salt concentration of 20 mM more realistically mimics the intracellular conditions. Under these conditions, our data show that at least two cytochrome c molecules bind simultaneously to the supercomplex surface. Additionally, previously unresolved residues at the N and C termini of subunits Qcr6 and Qcr9, respectively, were observed.

The cytochrome c-supercomplex interactions were also studied at 20 and 150 mM monovalent salt in mitoplasts containing the supercomplex. Overall activity in mitoplasts was lower than in detergent-purified supercomplexes. The results show that supercomplex activity as a function of cytochrome c concentration was similar at 20 mM and 150 mM salt, contrasting the behavior observed in detergent-purified supercomplexes. This difference is explained by a shift in the rate-limiting step in membrane-bound supercomplexes.

The effect of ionic strength was further studied by measuring the supercomplex activity, both in solution and in mitoplasts, at increasing salt concentrations. Increasing ionic strength resulted in a monotonic decrease in cytochrome c affinity for the supercomplex, indicating a classical Debye-Hückel behavior that contrasts earlier studies with non-biological systems. 

Finally, the structure of the Mycobacterium smegmatis supercomplex was resolved in native membranes, revealing a physical association with the enzyme malate:quinone oxidoreductase. Spectrophotometric analyses showed that malate:quinone oxidoreductase can transfer electrons from malate to the supercomplex, suggesting a connection between the Krebs cycle and aerobic respiration in mycobacteria.