ROS production by cytochrome bc: Its mechanism as inferred from the effects of heme b cofactor mutants.

Cytochrome bc is one of the enzymes of electron transport chain responsible for generation of reactive oxygen species (ROS). While ROS are considered to be products of side reactions of quinol oxidation site (Q), molecular aspects of their generation remain unclear. One of them concerns significance of hemes b (b and b) redox potentials (E) and properties on ROS generation by Q.

Identification of hydrogen bonding network for proton transfer at the quinol oxidation site of Rhodobacter capsulatus cytochrome bc.

Cytochrome bc catalyzes electron transfer from quinol (QH) to cytochrome c in reactions coupled to proton translocation across the energy-conserving membrane. Energetic efficiency of the catalytic cycle is secured by a two-electron and two-proton bifurcation reaction leading to oxidation of QH and reduction of the Rieske cluster and heme b. The proton paths associated with this reaction remain elusive.

On the inter-monomer electron transfer in cytochrome bc.

Cytochrome bc is a structural and functional homodimer. The catalytically-relevant inter-monomer electron transfer has been implicated by a number of experiments, including those based on analyses of the cross-dimer mutated derivatives. As some of the original data on these derivatives have recently been questioned, we extend kinetic analysis of these mutants to confirm the enzymatic origin of the observed activities and their relevance in exploration of conditions that expose electron transfer between the monomers.

Redox Active Antimicrobial Peptides in Controlling Growth of Microorganisms at Body Barriers.

Epithelia in the skin, gut and other environmentally exposed organs display a variety of mechanisms to control microbial communities and limit potential pathogenic microbial invasion. Naturally occurring antimicrobial proteins/peptides and their synthetic derivatives (here collectively referred to as AMPs) reinforce the antimicrobial barrier function of epithelial cells. Understanding how these AMPs are functionally regulated may be important for new therapeutic approaches to combat microbial infections.

Catalytic Reactions and Energy Conservation in the Cytochrome and Complexes of Energy-Transducing Membranes.

This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome and (Cyt/) membranous multisubunit homodimeric complexes. These remarkable molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient, which powers cellular metabolism in the majority of living organisms. Cyts/ share many similarities but also have significant differences.

ROS signaling capacity of cytochrome bc: Opposing effects of adaptive and pathogenic mitochondrial mutations.

Cytochrome bc, also known as mitochondrial complex III, is considered to be one of the important producers of reactive oxygen species (ROS) in living organisms. Under physiological conditions, a certain level of ROS produced by mitochondrial electron transport chain (ETC) might be beneficial and take part in cellular signaling. However, elevated levels of ROS might exhibit negative effects, resulting in cellular damage.

The antimicrobial activity of chemerin-derived peptide p4 requires oxidative conditions.

Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein abundantly produced in the skin epidermis. Despite the fact that most of the bactericidal activity present in human skin exudates is chemerin-dependent, just how chemerin shapes skin defenses remains obscure. Here we demonstrate that p4, a potent antimicrobial human chemerin peptide derivative, displays killing activity against pathogenic methicillin-resistant strains and suppresses microbial growth in a topical skin infection model.

Suppression of superoxide production by a spin-spin coupling between semiquinone and the Rieske cluster in cytochrome bc.

Catalytic reactions of quinol oxidoreductases may lead to the generation of superoxide due to electron leaks from unstable semiquinone intermediates (SQ). For cytochrome bc , the mechanism of suppression of superoxide generation remains unknown. We analyzed conditions of formation of a spin-spin-coupled state between SQ and the Rieske cluster (SQ-FeS) associated with catalysis of the quinol oxidation site of cytochrome bc .

Functional flexibility of electron flow between quinol oxidation Q site of cytochrome bc and cytochrome c revealed by combinatory effects of mutations in cytochrome b, iron-sulfur protein and cytochrome c.

Transfer of electron from quinol to cytochrome c is an integral part of catalytic cycle of cytochrome bc. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Q site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards cytochrome c, iii) reduction of cytochrome c by reduced [2Fe-2S] cluster, iv) reduction of cytochrome c by cytochrome c. In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, cytochrome c and cytochrome c.

[Molecular effects of mitochondrial mutations in cytochrome b of complex III and their impact on the levels of free radical production].

Cytochrome bc (mitochondrial complex III) is a common element of several bioenergetic systems. This enzyme catalyses electron transfer from ubiquinol to cytochrome c coupled to translocation of protons across the membrane, which contributes to generation of protonmotive force utilized for ATP production. Cytochrome b, together with cytochrome c and iron-sulfur protein (ISP), forms the evolutionarily conserved catalytic core.

Mitochondrial disease-related mutations at the cytochrome b-iron-sulfur protein (ISP) interface: Molecular effects on the large-scale motion of ISP and superoxide generation studied in Rhodobacter capsulatus cytochrome bc1.

One of the important elements of operation of cytochrome bc1 (mitochondrial respiratory complex III) is a large scale movement of the head domain of iron-sulfur protein (ISP-HD), which connects the quinol oxidation site (Qo) located within the cytochrome b, with the outermost heme c(1) of cytochrome c(1). Several mitochondrial disease-related mutations in cytochrome b are located at the cytochrome b-ISP-HD interface, thus their molecular effects can be associated with altered motion of ISP-HD. Using purple bacterial model, we recently showed that one of such mutations - G167P shifts the equilibrium position of ISP-HD towards positions remote from the Qo site as compared to the native enzyme [Borek et al.

Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer.

In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemesb The hemes display a large difference in redox midpoint potentials (ΔEm_b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on hemebligand mutants of cytochromebc1in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔEm_b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔEm_b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functionalin vivo This reveals that cytochromebc1can accommodate large changes in ΔEm_b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product.

Effect of H bond removal and changes in the position of the iron-sulphur head domain on the spin-lattice relaxation properties of the [2Fe-2S](2+) Rieske cluster in cytochrome bc(1).

Here, comparative electron spin-lattice relaxation studies of the 2Fe-2S iron-sulphur (Fe-S) cluster embedded in a large membrane protein complex - cytochrome bc1 - are reported. Structural modifications of the local environment alone (mutations S158A and Y160W removing specific H bonds between Fe-S and amino acid side chains) or in combination with changes in global protein conformation (mutations/inhibitors changing the position of the Fe-S binding domain within the protein complex) resulted in different redox potentials as well as g-, g-strain and the relaxation rates (T1(-1)) for the Fe-S cluster. The relaxation rates for T < 25 K were measured directly by inversion recovery, while for T > 60 K they were deduced from simulation of continuous wave EPR spectra of the cluster using a model that included anisotropy of Lorentzian broadening.

Mitochondrial Disease-related Mutation G167P in Cytochrome b of Rhodobacter capsulatus Cytochrome bc1 (S151P in Human) Affects the Equilibrium Distribution of [2Fe-2S] Cluster and Generation of Superoxide.

Cytochrome bc1 is one of the key enzymes of many bioenergetic systems. Its operation involves a large scale movement of a head domain of iron-sulfur protein (ISP-HD), which functionally connects the catalytic quinol oxidation Qo site in cytochrome b with cytochrome c1. The Qo site under certain conditions can generate reactive oxygen species in the reaction scheme depending on the actual position of ISP-HD in respect to the Qo site.

Catalytically-relevant electron transfer between two hemes bL in the hybrid cytochrome bc1-like complex containing a fusion of Rhodobacter sphaeroides and capsulatus cytochromes b.

To address mechanistic questions about the functioning of dimeric cytochrome bc1 new genetic approaches have recently been developed. They were specifically designed to enable construction of asymmetrically-mutated variants suitable for functional studies. One approach exploited a fusion of two cytochromes b that replaced the separate subunits in the dimer.

Enzymatic activities of isolated cytochrome bc₁-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains.

Homodimeric structure of cytochrome bc₁, a common component of biological energy conversion systems, builds in four catalytic quinone oxidation/reduction sites and four chains of cofactors (branches) that, connected by a centrally located bridge, form a symmetric H-shaped electron transfer system. The mechanism of operation of this complex system is under constant debate. Here, we report on isolation and enzymatic examination of cytochrome bc₁-like complexes containing fused cytochrome b subunits in which asymmetrically introduced mutations inactivated individual branches in various combinations.

Fusing two cytochromes b of Rhodobacter capsulatus cytochrome bc1 using various linkers defines a set of protein templates for asymmetric mutagenesis.

Cytochrome bc(1) (mitochondrial complex III), one of the key enzymes of biological energy conversion, is a functional homodimer in which each monomer contains three catalytic subunits: cytochrome c(1), the iron-sulfur subunit and cytochrome b. The latter is composed of eight transmembrane α-helices which, in duplicate, form a hydrophobic core of a dimer. We show that two cytochromes b can be fused into one 16-helical subunit using a number of different peptide linkers that vary in length but all connect the C-terminus of one cytochrome with the N-terminus of the other.

An electronic bus bar lies in the core of cytochrome bc1.

The ubiquinol-cytochrome c oxidoreductases, central to cellular respiration and photosynthesis, are homodimers. High symmetry has frustrated resolution of whether cross-dimer interactions are functionally important. This has resulted in a proliferation of contradictory models.

Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc₁. Implications for the mechanism of superoxide production.

In addition to its bioenergetic function of building up proton motive force, cytochrome bc₁ can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQ(o)) formed at the quinone oxidation/reduction Q(o) site (Q(o)) as a result of single-electron oxidation of quinol by the iron-sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b(L) (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist.

Movement of the iron-sulfur head domain of cytochrome bc(1) transiently opens the catalytic Q(o) site for reaction with oxygen.

Cytochrome bc(1), a key enzyme of biological energy conversion, generates or uses a proton motive force through the Q cycle that operates within the two chains of cofactors that embed two catalytic quinone oxidation/reduction sites, the Q(o) site and the Q(i) site. The Q(o) site relies on the joint action of two cofactors, the iron-sulfur (FeS) cluster and heme b(L). Side reactions of the Q cycle involve a generation of superoxide which is commonly thought to be a product of an oxidation of a highly unstable semiquinone formed in the Q(o) site (SQ(o)), but the overall mechanism of superoxide generation remains poorly understood.

Demonstration of short-lived complexes of cytochrome c with cytochrome bc1 by EPR spectroscopy: implications for the mechanism of interprotein electron transfer.

One of the steps of a common pathway for biological energy conversion involves electron transfer between cytochrome c and cytochrome bc1. To clarify the mechanism of this reaction, we examined the structural association of those two proteins using the electron transfer-independent electron paramagnetic resonance (EPR) techniques. Drawing on the differences in the continuous wave EPR spectra and saturation recoveries of spin-labeled bacterial and mitochondrial cytochromes c recorded in the absence and presence of bacterial cytochrome bc1, we have exposed a time scale of dynamic equilibrium between the bound and the free state of cytochrome c at various ionic strengths.