Brief encounters of cytochrome c

Abstract

Transient protein interactions are paramount to life where fast and efficient transfer of information and cargo are often integral to pathways and networks. However, complexes formed by transient protein interactions are often times resistant to direct structural characterization due to their inherent, dynamic nature, so our knowledge to date typically derives from biochemical, biophysical and computational methods. In this issue, Shimada and co‐authors present the crystal structure of the mammalian cytochrome c oxidase in complex with its electron donor cytochrome c, identifying a new class of protein–protein interaction termed “soft and specific”.

Cytochrome c oxidase (Complex IV, CcO) is a member of the haem copper oxidase superfamily (HCO), which functions as the terminal enzyme in the respiratory chain of mitochondria and aerobic prokaryotes (Ferguson‐Miller & Babcock, 1996). It couples the reduction in molecular oxygen to transmembrane proton pumping driving downstream adenosine triphosphate (ATP) biosynthesis. The mechanisms governing the electron and proton transfer reactions in the individual respiratory complexes have been areas of study for decades.

It has been over 20 years since Tsukihara et al reported the first structures of the dimeric mammalian cytochrome c oxidase (Tsukihara et al, 1995, 1996). Since then, significant progress has been made on the structure determination of the individual respiratory complexes (Complexes I–IV) and the elucidation of the spatial organization and stoichiometries of these complexes in respiratory supercomplexes, the so‐called respirasomes, through which they perform their various electron transfer and proton translocation functions (Sousa et al, 2016). However, the underlying mechanism behind the subsequent transfer of electrons from the soluble electron carrier cytochrome c (Cyt.c) to cytochrome c oxidase has remained elusive, with interaction models proposed based on extensive biochemical, solution NMR and molecular docking simulations.

Building on recent electron crystallography studies on two‐dimensional crystals of the Cyt.c–CcO complex (Osuda et al, 2016), the authors obtained and optimized three‐dimensional crystals of the complex under high pH conditions (Shimada et al, 2017). The resulting high‐resolution crystal structure of the Cyt.c–CcO complex offers the first view of the transient inter‐protein interactions involved in the complex and allowed the authors to propose an electron transfer pathway between the donor and acceptor. This pathway consists of a combination of through‐bond and through‐space jumps, and is markedly different from that of the covalent Cyt.c–CcO complex found in the structure of the thermophilic caa ₃‐type cytochrome oxidase, with only the transfer to the CuA binuclear centre from the axial methionine conserved (Lyons et al, 2012).

While molecular docking simulations and NMR studies had previously predicted a number of the inter‐protein interactions identified in the complex, unexpected features of the X‐ray structure highlight a number of differences relating to the nature of the contact interface and its size. Furthermore, comparison of the Cyt.c–CcO complex with reported protein–protein complexes, including other reported electron transport complexes, demonstrated that the minimal backbone distance between both proteins is significantly larger for the reported Cyt.c–CcO complex. This large inter‐protein span results in a small interfacial surface, with mutual contact between a limited number of long flexible polar residues, that orient cytochrome c, and the presence of three water layers with various interacting modes that function as a cushion between the proteins, facilitating the observed dynamic nature of Cyt.c with respect to CcO (Fig 1). The authors describe this novel protein interaction mode as “soft and specific contact”.

Figure 1. Comparison of the Cyt.c mediated electron transfer complexes of Complex IV and Complex III.

(A, B) Architecture of the electron transfer complex between (A) Complex IV and Cyt.c (Shimada et al, 2017), with (B) a detailed view of highlighted region rotated by ~120°. (C) For comparison, the Complex III–Cyt.c (PDB ID: 3cx5) is shown. Cytochrome c is depicted in orange, while the interacting subunits, SUII in Complex IV and SUIV in Complex III, are depicted as grey surfaces in (B) and (C), respectively. Interfacial waters forming the “soft and specific” binding site are indicated as yellow spheres with non‐interacting waters as red non‐bonded spheres.

Interesting, the same face of cytochrome c interacts through a repeated association/dissociation with both Complex III and Complex IV, meaning that electrons are accepted and donated by the same site in Cyt.c thus indicating that the binding modes in both complexes have evolved to carry out their respective specialized functions. The functional implications of the reported inter‐protein interaction likely serve to facilitate the fast on and off rates observed for Cyt.c through minimizing the energy barrier required for electron transfer complex formation and relaxing the need for dehydration at the interfacial area, as reported for the complex between Complex III and Cyt.c (Fig 1C) (Solmaz & Hunte, 2008).

What comes next? Although excellent, the high‐resolution Cyt.c–CcO complex reported here offers an initial view of electron transfer. Clearly, additional structural and functional characterization and biophysical modelling of these electron transfer complexes are required to further unravel the mechanism of energy transduction, and the discrete bioenergetic implications of the various binding modes of cytochrome c. Is this an encounter complex or the actual electron transfer interaction? The under‐representation of structures of these transient protein complexes belies their functional importance, and is likely a reflection of the challenge in obtaining suitable crystals for X‐ray diffraction studies. The recent emergence of high‐resolution single‐particle cryo‐electron microscopy with new possibilities for analysis of heterogenous samples may greatly accelerate the identification of such elusive “soft and specific” interactions.

See also: S Shimada et al (February 2017)