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Living organisms derive most of their free energy from oxidation-reduction (redox) reactions, i.e. processes involving transfer of electrons. To accomplish this, they use enzymes, oxidoreductases, which catalyze individual chemical redox reactions. These enzymes embed redox active metal cofactors, such as hemes and iron-sulfur clusters, and are often assembled into chains that perform specific biological functions. How these systems are designed to secure both the recognition of the reacting partners and catalysis is central to our understanding of the nature of their energetic efficiency and physiological regulation. It now becomes clear that more or less severe dysfunctions of bioenergetic enzymes are associated with increased vulnerability to many types of side reactions, including those commonly associated with production of damaging reactive oxygen species (ROS). This can create conditions predisposing towards or resulting in various diseases, such as several neurodegenerative and metabolic disorders, aging and aging-related diseases, and cancer.

We focus on the ubiquitous group of metalloproteins consisting of multi-subunit complexes responsible for oxidation/reduction of quinol/quinone, the evolutionary-conserved reactions that contribute to generation of the protonmotive force necessary for production of ATP. Specific enzymes which are of interest to us include mitochondrial complex III, its bacterial homologue - cytochrome bc1, plant cytochrome b6f and alternative complex III fulfilling the function of cytochrome bc1 in some bacteria.

We comprehensively investigate these proteins at molecular and sub-molecular levels to understand the mechanism of operation of the catalytic sites, the thermodynamic basis of the involvement of the metal centres, engineering of electron and proton transfers within and between the catalytic sites, the dynamics of macromolecular interactions with substrates and redox-protein partners (such as cytochrome c, plastocyanin, ferredoxin, FNR), the physiological aspects of regulation of respiratory and photosynthetic electron flow and engagement in redox signalling. Our conceptual framework considers both the energy-conserving catalytic reactions, as well as the energy-wasting short-circuits or leaks of electrons and, associated with them, the generation of ROS. Bacterial models are exploited for genetic manipulations to target specific structural elements for functional studies. This includes mimicking mitochondrial disease-related or adaptive mutations to unravel their molecular effects and modifying proteins for site-directed spin-labelling used to probe various molecular interactions and conformational dynamics.

[Physiol. Rev. (2015) 95, 219-243; Chemical Rev. (2021) 121, 2020-2108]


  • Using spin-labeled cytochrome c, we defined short (submillisecond) lifetime of the complex formed between cytochrome bc1 and its redox partner – cytochrome c. This implicated a diffusion-coupled mechanism of interaction in which the inter-protein electron transfer is a product of several collisions between the interacting proteins. This mechanism is relevant to other protein-protein interactions in redox systems
    [Metallomics (2011) 3, 404-409; J. Phys. Chem. B (2014) 118, 6634-6643] 

  • Using an unprecedented fusion of two monomeric cytochrome b subunits of cytochrome bc1 we broke the inherent symmetry of this enzyme by constructing several asymmetrically mutated variants of this enzyme to reveal occurrence of catalytically-relevant electron transfer between the monomers. This implied that the enzyme forms an unusual H-shaped electron transfer system that connects all four catalytic quinone binding sites of the dimer. While the physiological significance of the H-shape design remains unclear, possible advantages of it are considered in the context of regulation of electron flow, increased target size for substrates, mechanisms of suppression of ROS production, protection against damaging effects of mitochondrial mutations that accumulate with age or under oxidative stress (it is reasonable to assume that it builds in redundancy to allow physiological function of the enzyme after operational damage of one part).
    [Science (2010) 329, 451-454; Biochemistry (2012) 51, 829-835; Biochim. Biophys. Acta (2013) 1827, 751-760]

  • Using selectively modified chains of cytochrome bc1, we identified molecular conditions of generation of superoxide by the quinol oxidation site (the Qo site) of cytochrome bc1. We formulated a new molecular mechanism (“Semireverse – Rieske off”) which proposes that flow of electron back to the Qo site significantly increases probability of reaction of semiquinone with molecular oxygen when one of the cofactors (the Rieske cluster) is temporarily absent from the site (this absence is a consequence of the movement of the head domain containing the cluster (ISP-HD) between the Qo site and the outermost cofactor, heme c1, that naturally occurs during the catalytic cycle). This model puts forward the idea that under the conditions of impeded electron flow, the short-circuit reactions effectively compete with leaks on oxygen to compromise the energy wasting formation of damaging ROS with the energy-wasting but leak-proof short circuits. This model also allows us to understand some of the specific molecular effects observed in the bacterial mimics of mitochondrial disease-related mutations.
    [Biochemistry (2008) 47, 12365-12370;  Biochim. Biophys. Acta (2010) 1797, 1820-1827;  J. Biol. Chem. (2015) 290, 23781-23792; Biochim. Biophys. Acta (2016) 1857, 1102-1110; Nature Comm. (2020) 11: 322; Free Rad. Biol. Med. (2021) 163, 243-254]

  • Using low-temperature EPR, we discovered a new metastable radical intermediate associated with operation of the Qo site of cytochrome bc1 and cytochrome b6f. This intermediate is a spin-coupled state of the semiquinone and the reduced Rieske cluster (the SQ-FeS state). We proposed a mechanism in which the SQ-FeS state safely hold electrons at a local energetic minimum, becoming possible element of regulation: it might serve as a ‘buffer’ for electrons that are unable to be relegated from the site upon progress of catalytic reactions. We formulated a general  model in which modulation of energy level of the SQ-FeS state in the energy landscape in photosynthetic vs respiratory enzymes provides mechanism to adjust electron transfer rates for efficient catalysis under different oxygen tensions. We also proposed that SQ-FeS suppresses superoxide generation, becoming an element modulating superoxide release under physiologically-relevant conditions slowing electron flow through the enzyme.
    [Biochemistry (2013) 52, 6388-6395; Proc. Natl Acad. Sci. USA (2017) 114, 1323-1328; Biochim. Biophys. Acta (2018) 1859, 145-153; FEBS Lett. (2019) 593, 3-12]

  • Using mutational studies, low-temperature EPR and quantum mechanical calculations we explained how the catalytic site of cytochrome bc1 promotes quinone reduction during which two electrons are transferred sequentially and the stable semiquinone is an intermediate step of the reaction. We proposed a mechanism in which the specific charge and spin polarization imposed by the catalytic site upon formation of semiquinone is a key element of control of the proper sequence of electron and proton transfers. We proposed that this mechanism may also be applicable to other quinone oxidoreductases.
    [Biochim. Biophys. Acta (2020) 1861, 148216]

  • Using mutational studies, low-temperature EPR and molecular dynamics simulations we described molecular factors that govern the electrochemical properties of hemes in redox-active proteins. We observed unexpected molecular effects of mitochondrial disease-associated mutation on the properties of one of the hemes of cytochrome bc1. These effects revealed that stabilization of the specific hydrogen bond to heme propionate on one hand increases the redox midpoint potential, but on the other, destabilizes the low spin state of the oxidized heme. This exposed a dual role of this bond pointing out toward a necessity of the careful adjustment of the strength of this bond to secure a proper redox potential and a proper spin state of the heme. We formulated a principle that might apply for other heme proteins and, possibly, be exploited in engineering of artificial heme-containing redox systems.
    [Proc. Natl Acad. Sci. USA (2021) 118, e2026169118]