Análisis e interpretación de las encuestas a los trabajadores

Currently, the molecular structures of human mitochondrial proteins have not yet been determined; as such protein structures from species closely related to human (sharing protein homology of over 70%) were used to identify the location of specific mutations. These proteins were identified through the Protein Data Bank (PDB) database, and the structures were downloaded from The Research Collaboratory for Structural Bioinformatics (RCSB) PDB database to be examined on the Swiss-PDB viewer (v.4.1.0) software. Only protein complexes III and IV of the human ETC were examined, since the bovine protein counterparts for these complexes were the only structures available that were over 70% homologous to the human protein complexes.

Before inferences can be drawn from these protein models, on the effect of any non-synonymous mutations, it was necessary to understand the interactions between different protein subunits and how these work together to create a working protein complex.

1.5.4.1.1. Cytochrome bc1 complex

The cytochrome bc1 complex represents Complex III of the mitochondrial

processes from the hydrophobic carrier ubiquinol to cytochrome c, during which there is a redox reaction that is initiated from the oxidation of hydroquinones (QH2) to quinones (Q). This is followed by the reduction of cytochrome c 87.

Figure 1.8 Diagram of the Complex III monomer, cytochrome bc1. The locations of the redox prosthetic groups in cytochrome b (heme bL and bH) and in cytochrome c1 (heme c1) are shown. The main processes of the Q cycle are also represented, in addition to the net movement of the protons during catalysis.

Interaction between cytochromes b and c1 forms the cytochrome bc1 core

complex. However, completion of the structure requires the presence of the Rieske iron-sulfur protein, which spans cytochrome b and c1 thereby connecting

the 2 monomers88. The iron-sulfur cluster resides within the mobile C-terminus globular domain of the Rieske iron-sulfur protein89_{. Stabilisation of this redox}

prosthetic group occurs only after the protein has formed a complete complex with cytochrome bc189. Formation of the cytochrome bc1 complex is made

possible as the c-terminal helix of cytochrome c1 interacts with the fifth

cytochrome b helix. The flexibility of the Fe/S protein head domain permits interaction with the cytochrome bc1 complex87, which is essential as the

frequency at which the head domain moves controls the rates of catalysis90.

The Q cycle is responsible for carrying out the functions of cytochrome bc1

complex. Two catalytic sites exist that allow for oxidation of ubihydroquinone (QH2) and reduction of ubiquinone to ubiquinol (Qi). The Qo site and Qi site

reside opposite one another on the positive and negative side of the inner membrane, respectively91. Although the two catalytic centers Qi and Qo are

located apart from one another, they are actively coupled to each other88.

During two consecutive oxidation reactions of QH2 at the Qo site, two electrons

are lost. One electron is transferred to the Qi site to re-generate ubihydroquinone

(QH2), which is then released from the enzyme complex92. The other electron is

shuttled to the 2Fe2S cluster found in the Fe-S protein. This second electron is transferred from the Fe-S protein to cytochrome c1 during catalysis by movement

of the head domain, from which it is further transferred to the hydrophobic carrier, cytochrome c, and shuttled to cytochrome c oxidase (Complex IV). During the Q cycle, by-products of superoxides can be formed from the oxidation of ubiquinol, as a result of electron leakage93. Nonetheless, the primary function of the Q cycle is in the generation of a membrane potential across the mitochondrial inner membrane. The net yield of 2 protons per catalytic cycle

contributes towards a proton gradient87, which is important for the production of ATP via ATP synthase (Complex V)92.

1.5.4.1.2. Cytochrome c oxidase complex

Cytochrome c oxidase (COX) I-III together form the catalytic core of Complex IV94 (Figure 1.9). Within the hydrophobic core of COX I, there are two heme A cofactors (a and a3) 95. The flow of electrons begins from the hydrophobic carrier cytochrome c, which transfers electrons to the CuA centre in the COX II subunit,

and subsequently to the heme a site. From here, electrons are shuttled to the heme a3-CuB bimetallic centre, where oxygen binds and becomes reduced to

water96.

  Figure 1.9 Cytochrome c oxidase (Complex IV) of the mitochondrial ETC. Three subunits (COX I, II and III) comprise the catalytic core of Complex IV, with the two heme A cofactors, the copper centres and the reduction of oxygen to water.

COX II is highly hydrophilic compared to COX I and III97, and is structurally the smallest of all the subunits that comprise the catalytic core of cytochrome c oxidase98. On the other hand, COX III is absent of any prosthetic groups, with its structure being defined by the presence of seven membrane-spanning alpha helices.

Assembly of Complex IV occurs in a successive manner, initiating from COX I followed closely by recruitment of accessory factors such as heme groups, and subsequently by other subunits99. As a result, defects arising early during this process are thought to have a cumulative effect on the overall assembly, and thereby functionality of the protein complex95. Following the complete assembly of Complex IV, there appears to be no interaction between COX II and III, as they interact only through the transmembrane region of COX I 100.

Quality control regarding assembly of the proteins occurs throughout protein biosynthesis, at both the subunit level and also when the complex is fully formed94. However, since there is thought to be a mutation threshold level, some defects in the mitochondrial respiratory chain are permitted, as non-mutated counterparts are believed to compensate for activity in the mutant proteins95.

All subunits of Complex IV together with cytochrome B represent the mtDNA- encoded proteins that are most conserved throughout evolution. COX I is the most conserved region (47.2%) amongst the 13 mtDNA-encoded polypeptides, when comparing the human amino acid sequences to the yeast protein counterpart. This was followed by cytochrome B (32.4% protein homology) and

the remaining Complex IV subunits COX III (30.8% protein homology) and COX II (28.6% protein homology)101. Reasons for protein conservation for these mtDNA regions can be attributed to their roles as key components of the ETC. In summary, cytochrome B has the important role of catalysing the transfer of electrons from the hydrophobic carrier ubiquinol to cytochrome c, which is coupled to the translocation of protons across the inner membrane of the mitochondrion. Within the cytochrome bc1 complex, there are prosthetic groups

and redox centres that collectively maintain activity of the Q cycle102. Complex IV catalyses the final stages of the ETC by reducing molecular oxygen to water, and as such, mutations to the complex subunits are more likely to be less tolerable. For example, the region within COX II that is involved with the shuttling of electrons from cytochrome c to the CuA center has been reported to

be very highly conserved102. Consequently, mtDNA variants detected within these regions have a greater chance of being predicted to cause protein impairment.