Loss of a Conserved Tyrosine Residue of Cytochrome b Induces Reactive Oxygen Species Production by Cytochrome bc1

Abstract

Production of reactive oxygen species (ROS) induces oxidative damages, decreases cellular energy conversion efficiencies, and induces metabolic diseases in humans. During respiration, cytochrome bc 1 efficiently oxidizes hydroquinone to quinone, but how it performs this reaction without any leak of electrons to O 2 to yield ROS is not understood. Using the bacterial enzyme, here we show that a conserved Tyr residue of the cytochrome b subunit of cytochrome bc 1 is critical for this process. Substitution of this residue with other amino acids decreases cytochrome bc 1 activity and enhances ROS production. Moreover, the Tyr to Cys mutation cross-links together the cytochrome b and iron-sulfur subunits and renders the bacterial enzyme sensitive to O 2 by oxidative disruption of its catalytic [2Fe-2S] cluster. Hence, this Tyr residue is essential in controlling unproductive encounters between O 2 and catalytic intermediates at the quinol oxidation site of cytochrome bc 1 to prevent ROS generation. Remarkably, the same Tyr to Cys mutation is encountered in humans with mitochondrial disorders and in Plasmodium species that are resistant to the anti-malarial drug atovaquone. These findings illustrate the harmful consequences of this mutation in human diseases.In most organisms, the ubiquinol:cytochrome c oxidoreductase (cytochrome bc 1 or complex III) is a central enzyme for ATP production through oxidative phosphorylation that relies on the proton motive force (⌬H ϩ ) generated by the respiratory chain (1). Production of reactive oxygen species (ROS) leads oxidative damages of cellular components and in eukaryotes induces apoptosis (2, 3). Most cellular ROS are thought to emanate from the respiratory NADH:dehydrogenase (i.e. complex I) and cytochrome bc 1 (see Fig. 1a for Rhodobacter capsulatus structure) under compromising physiological conditions (4, 5). Cells use antioxidant enzymes (e.g. superoxide dismutase or glutathione peroxidase) to prevent oxidative damages, but upon extensive ROS generation, harmful damage occurs. Indeed, cellular redox homeostasis, regulated by the rate of electron flow through the respiratory chain and O 2 availability, is tightly coupled with the global metabolism (4, 5). For example, recent studies show that cytochrome bc 1 is involved in stabilization and activation of hypoxia-induced factors like HIF-1␣ by mitochondria-generated ROS under hypoxic conditions (6, 7).Mitochondrial DNA mutations are known causes of clinical syndromes (e.g. LHON, Pearson syndrome, and exercise intolerance) or provide predisposition for inherited and common diseases (e.g. aging, cardiomyopathy, cancer, diabetes, and neurodegenerative diseases) (8, 9). Mutations in mitochondrial or nuclear DNAs associated with mitochondrial fission and fusion (10), ascribed to ROS generation, lead to progressive dysfunction of mitochondria and loss of energy efficiency (11). For example, the Tyr to Cys mutation at position 278 (position 302 in R. capsulatus; see Fig. 1b) of cytochrome b of human mitochondrial cytochrome bc 1...

Saccharomyces cerevisiae-Based Mutational Analysis of the bc 1 Complex Q o Site Residue 279 To Study the Trade-Off between Atovaquone Resistance and Function

“…However, that hydrogen bond seems to have only a modest role since the replacement of tyrosine by phenylalanine has little effect on the bc 1 complex function as shown in Table 1, a finding which is in agreement with previous studies using bacterial enzymes (25,27).…”

“…It was previously shown that Y279A, C, and S mutant enzymes exhibited a low SO production, a finding very similar to that observed here (24). Similar observations were reported using Rhodobacter capsulatus Y279 mutants (25). The increase in SO production can be understood in terms of higher semiquinone occupancy at the Q o site, increasing the equilibrium concentration of semiquinone for reaction with oxygen or an increased rate constant for the reaction with oxygen, perhaps by steric means (i.e., an increased probability of collision with oxygen).…”

“…In bacteria, analysis of mutant bc 1 complexes showed that replacement of Y279 by valine or leucine had a moderate effect on the enzyme activity, that alanine and cysteine increased the severity, and that serine and glycine caused further activity loss. The most severely impaired mutant seemed to be Y279Q (25,27). It thus appears that, in yeasts as in bacterial complexes, the severity of the effect is determined by both the size and the polarity of the side chain.…”

Putative 3D Structure of QcrB from Mycobacterium tuberculosis Cytochrome bc1 Complex, a Novel Drug‐Target for New Series of Antituberculosis Agent Q203

“…In human, mutation of this residue causes severe exercise intolerance and "multisystem disorder". 26 In addition, it is known that Tyr268 mutated into Ser in malarial parasite occurred resistance to the antimalarial drug atovaquone. 27 It is also important for catalytic activity of the enzyme in bacteria and yeast.…”

Interplay between the hinge region of iron sulphur protein and the Qo site in the bc1 complex — Analysis of Plasmodium-like mutations in the yeast enzyme

“…The positioning of the residue is proposed to be governed by displacement of the PEWY loop (the 'PEWY seesaw') and the docking of the ISP headgroup at the Q o site [30,31]. This proposed 'trapdoor' mechanism is likely to have a role in preventing side-reactions and SO production by minimising semiquinone occupancy at Q o site, a proposal supported by the observation of increased ROS production in mutants equivalent to Y279 in Rhodobacter capsulatus bc 1 complex [32]. It may be postulated that a phenylalanine, having perhaps more degrees of freedom of movement due to the lack of a potentially hydrogen-bonding hydroxyl moiety, is a more efficient trapdoor for the Plasmodium-like Q o site.…”