Binding Site For Cytochrome Research Articles

Molecular characterization of a complex of apoptosis-inducing factor 1 with cytochrome c oxidase of the mitochondrial respiratory chain

Combining mass spectrometry-based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis-inducing factor 1 (AIFM1) forms a defined complex with ∼10% of monomeric cytochrome c oxidase (COX) but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 intercross-links engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM12 complex (PDBDEV_00000092). An application of two complementary proteomic approaches thus provided unexpected insight into the macromolecular organization of the mitochondrial complexome. Our structural model excludes direct electron transfer between AIFM1 and COX. Notably, however, the binding site of cytochrome c remains accessible, allowing formation of a ternary complex. The discovery of the previously overlooked COX-AIFM12 complex and clues provided by the structural model hint at potential roles of AIFM1 in oxidative phosphorylation biogenesis and in programmed cell death.

Fine Tuning of Real Time PCR as a First Tool for the Detection of G143A Substitution in <i>Venturia inaequalis</i> Samples

Apple scab caused by Venturia inaequalis (Cke.) Wint. is the most important disease of apple trees worldwide and requires a high number of fungicide applications. The G143A substitution in the inhibitor binding site of cytochrome b of V. inaequalis confers a high level of resistance to strobilurins targeting the bc1 complex. The aim of this work was to substitute the labor intensive in vitro assays, with the faster quantitative PCR. An allele-specific qPCR method with a newly designed primer set was successfully developed to quantitatively determine the frequency of QoI-resistant A143 allele in populations of V. inaequalis. To be able to suggest that the molecular method could be applied as unique and robust technique, we carried out in vitro sensitivity test to trifloxystrobin; first testing the relative germination and subsequently confirmed with the quantification of mutated allele frequencies by qPCR on forty-nine Italian V. inaequalis populations. qPCR gave a similar pattern to that obtained using in vitro conidial germination test in predominantly sensitive and resistant populations, the variability between these two tests was observed in some heterogeneous populations. The qPCR assay developed in this study efficiently quantifies the A143 allele and we can conclude that this method could be useful for the study of the fungicide resistance at population level in the fields, giving a quick response also with a large amount of samples.

Characterization of the mechanism of action of the fungicide fenpicoxamid and its metabolite UK-2A.

BACKGROUNDFenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated.RESULTSUK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides.CONCLUSIONFenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cation Binding Site of cytochrome c oxidase: Progress report

Cytochrome c oxidase from bovine heart binds Ca2+ reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca2+ shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca2+-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the “slow” form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

Computational Prediction and in Vitro Analysis of Potential Physiological Ligands of the Bile Acid Binding Site in Cytochrome c Oxidase

A conserved bile acid site has been crystallographically defined in the membrane domain of mammalian and Rhodobacter sphaeroides cytochrome c oxidase (RsCcO). Diverse amphipathic ligands were shown previously to bind to this site and affect the electron transfer equilibrium between heme a and a3 cofactors by blocking the K proton uptake path. Current studies identify physiologically relevant ligands for the bile acid site using a novel three-pronged computational approach: ROCS comparison of ligand shape and electrostatics, SimSite3D comparison of ligand binding site features, and SLIDE screening of potential ligands by docking. Identified candidate ligands include steroids, nicotinamides, flavins, nucleotides, retinoic acid, and thyroid hormones, which are predicted to make key protein contacts with the residues involved in bile acid binding. In vitro oxygen consumption and ligand competition assays on RsCcO wildtype and its Glu101Ala mutant support regulatory activity and specificity of some of these ligands. An ATP analog and GDP inhibit RsCcO under low substrate conditions, while fusidic acid, cholesteryl hemisuccinate, retinoic acid, and T3 thyroid hormone are more potent inhibitors under both high and low substrate conditions. The sigmoidal kinetics of RsCcO inhibition in the presence of certain nucleotides is reminiscent of previously reported ATP inhibition of mammalian CcO, suggesting regulation involving the conserved core subunits of both mammalian and bacterial oxidases. Ligand binding to the bile acid site is noncompetitive with respect to cytochrome c and appears to arrest CcO in a semioxidized state with some resemblance to the "resting" state of the enzyme.

Blind Prediction of Charged Ligand Binding Affinities in a Model Binding Site

Predicting absolute protein–ligand binding affinities remains a frontier challenge in ligand discovery and design. This becomes more difficult when ionic interactions are involved because of the large opposing solvation and electrostatic attraction energies. In a blind test, we examined whether alchemical free-energy calculations could predict binding affinities of 14 charged and 5 neutral compounds previously untested as ligands for a cavity binding site in cytochrome c peroxidase. In this simplified site, polar and cationic ligands compete with solvent to interact with a buried aspartate. Predictions were tested by calorimetry, spectroscopy, and crystallography. Of the 15 compounds predicted to bind, 13 were experimentally confirmed, while 4 compounds were false negative predictions. Predictions had a root-mean-square error of 1.95kcal/mol to the experimental affinities, and predicted poses had an average RMSD of 1.7Å to the crystallographic poses. This test serves as a benchmark for these thermodynamically rigorous calculations at predicting binding affinities for charged compounds and gives insights into the existing sources of error, which are primarily electrostatic interactions inside proteins. Our experiments also provide a useful set of ionic binding affinities in a simplified system for testing new affinity prediction methods.

New Ligands of the Conserved Steroid Binding Site of Cytochrome c Oxidase

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain of mitochondria and many bacteria, requires the uptake of protons for activity via two pathways, the D-path and the K-path. Among the mutants of Rhodobacter sphaeroides (Rs) CcO that inhibit proton uptake in the K-path is E101A in subunit II, which removes a key carboxyl at the entrance to the pathway and decreases the activity of the purified enzyme over 20-fold. Micromolar levels of the bile acids cholate or deoxycholate were shown to stimulate E101A activity 10-fold, and crystals of wildtype RsCcO grown in the presence of deoxycholate (Qin et al., Biochemistry 47:9931-9933, 2008) showed a single deoxycholate molecule bound close to E101 in the same location as a cholate molecule observed in bovine CcO crystal structure (PDB 1OCC). Evidence of protective effects of steroids against CcO inhibition in bilirubin neurotoxicity and in Alzheimers (Vaz et al., J. Neurochem. 112:56-65, 2010; Tillement et al., Steroids 71:725-735, 2006) suggest that this site could be physiologically relevant to regulation of CcO.Here we use the RsCcO mutant E101A as a sensitive assay to further investigate the nature of this conserved site. Activity assays show that certain other steroids (cholesterol hemisuccinate) and lipidic molecules (phytanic acid, arachidonic acid) and some detergents affect RsCcO in a manner that indicates that these ligands also bind at the same site. The studies reveal a high degree of specificity and suggest possible physiological regulators. Crystallographic and computational approaches are underway to identify additional potential ligands and to determine the physiological significance of this conserved binding site. (NIH GM26916)

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O2 binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO2 through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO2. ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO2, but it was unaffected at low pO2 in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42 degrees C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O2 binding at CcO at complex IV.

Role of phospholipids in respiratory cytochrome bc1 complex catalysis and supercomplex formation

Specific protein–lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc 1 complex and how disruption of a specific cardiolipin binding site in cytochrome c 1 alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc 1 complex was treated with phospholipase A 2. The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from − 52 to − 82 mV (heme b L) and from + 113 to − 2 mV (heme b H). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CL i) in supercomplex formation by site-directed mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc 1 complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein–protein interaction.

Probing the recognition specificity of a protein molecularly imprinted polymer using force spectroscopy

Molecularly imprinted polymers (MIPs) are synthetic receptors obtained by polymerization using molecular templates. We have synthesized MIP films (co-polymers of acrylamide and different acrylic acid-based cross-linkers) with specific binding sites for cytochrome c, which were imprinted in the bulk or in the surface. The binding specificity of the polymers was studied at the macroscale by equilibrium binding experiments with fluorescein-labeled cytochrome c. Imprinting factors of up to 4.1 were obtained that were a function of the cross-linker used and the degree of cross-linking. We have then employed, for the first time, AFM force spectroscopy to directly measure the force of interaction of the protein with the synthetic receptor sites obtained by molecular imprinting. The polymer surfaces were scanned with AFM cantilevers carrying covalently attached cytochrome c molecules, giving raise to specific binding events with binding forces between 85 and 95pN. Control cantilevers without cytochrome c or with covalently attached bovine serum albumin, as well as non-imprinted control polymers, did not yield specific binding events. We believe that these results demonstrate the great potential of force spectroscopy for the characterization of molecularly imprinted polymers.

Engineering the Substrate Specificity and Reactivity of a Heme Protein: Creation of an Ascorbate Binding Site in Cytochrome c Peroxidase

The binding of substrates to heme enzymes has been widely assumed to occur at the so-called delta-heme edge. Recently, however, a number of examples have appeared in which substrate binding at an alternative site, the gamma-heme edge, is also possible. In previous work [Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307], we showed that binding of ascorbate to ascorbate peroxidase occurred at the gamma-heme edge. Here, we show that the closely related cytochrome c peroxidase enzyme can duplicate the substrate binding properties of ascorbate peroxidase through the introduction of relatively modest structural changes at Tyr36 and Asn184. Hence, crystallographic data for the Y36A/N184R/W191F triple variant of cytochrome c peroxidase shows ascorbate bound to the gamma-heme edge, with hydrogen bonds to the heme propionate and Arg184. In parallel mechanistic studies in variants incorporating the W191F mutation, we show that a transient porphyrin pi-cation radical in Compound I of cytochrome c peroxidase, analogous to that observed in ascorbate peroxidase, is competent for ascorbate oxidation but that under steady state conditions this intermediate decays too rapidly to sustain efficient turnover of ascorbate. The results are discussed in terms of our more general understanding of substrate oxidation across other heme proteins, and the emerging role of the heme propionates at the gamma-heme edge.

A Conserved Steroid Binding Site in Cytochrome c Oxidase

Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

The binding of cytochrome c to neuroglobin: A docking and surface plasmon resonance study

It has recently been proposed that the role of neuroglobin in the protection of neurons from ischaemia induced cell death requires the formation of a transient complex with cytochrome c. No such complex has yet been isolated. Here, we present the results of soft docking calculations, which indicate one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved. surface plasmon resonance studies provide a value of 45 μM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation is associated with a small unfavourable enthalpy change (1.9 kcal mol −1) and a moderately large, favourable entropy change (14.8 cal mol −1 deg −1). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions.

Rapid electrostatic evolution at the binding site for cytochrome c on cytochrome c oxidase in anthropoid primates

Cytochrome c (CYC) oxidase (COX), a multisubunit enzyme that functions in mitochondrial aerobic energy production, catalyzes the transfer of electrons from CYC to oxygen and participates in creating the electrochemical gradient used for ATP synthesis. Modeling three-dimensional structural data on COX and CYC reveals that 57 of the >1,500 COX residues can be implicated in binding CYC. Because of the functional importance of the transfer of electrons to oxygen, it might be expected that natural selection would drastically constrain amino acid replacement rates of CYC and COX. Instead, in anthropoid primates, although not in other mammals, CYC and COX show markedly accelerated amino acid replacement rates, with the COX acceleration being much greater at the positions that bind CYC than at those that do not. Specifically, in the anthropoid lineage descending from the last common ancestor of haplorhines (tarsiers and anthropoids) to that of anthropoids (New World monkeys and catarrhines) and that of catarrhines (Old World monkeys and apes, including humans), a minimum of 27 of the 57 COX amino acid residues that bind CYC were replaced, most frequently from electrostatically charged to noncharged residues. Of the COX charge-bearing residues involved in binding CYC, half (11 of 22) have been replaced with uncharged residues. CYC residues that interact with COX residues also frequently changed, but only two of the CYC changes altered charge. We suggest that reducing the electrostatic interaction between COX and CYC was part of the adaptive evolution underlying the emergence of anthropoid primates.

Spectroscopic studies on electron transfer between plastocyanin and cytochrome b6f complex

This paper reports the results of the research on the interaction between the highly active cytochrome b 6 f complex and plastocyanin, both isolated from the same source – spinachia oleracea plants. An equilibrium constant K between the cytochrome f of the cytochrome b 6 f complex and plastocyanin has been estimated by two independent spectroscopic techniques: steady-state absorption spectroscopy and stopped-flow. The second-order rate constants k 2 for forward and backward electron transfer between cytochrome f and plastocyanin have been found between 1.4–2 × 10 7 and 8–10 × 10 6 M −1 s −1, respectively, giving the value of an equilibrium constant of about 2 ± 0.4 or a difference in redox potential between plastocyanin and cytochrome f of cytochrome b 6 f complex of ca. 17 mV. The value of K=1.7±0.3 has been estimated from steady-state experiments in which the initial and final concentrations of participating components after mixing have been estimated via differential spectra analysis or spectra deconvolution. We propose a method of evaluation of the final plastocyanin concentration after the electron transfer reaction between cytochrome bf complex and plastocyanin that overcomes the interference by the strong chlorophyll absorption in the spectral region where oxidised plastocyanin has its low extinction absorption band. The data from both experiments, in the system devoid of quinol being the electron donor to cytochrome b 6, suggest that in case of electron transfer from cytochrome f to plastocyanin electron transfer can either bypass cytochrome f or the Rieske iron–sulfur protein can be reduced prior to its movement to the quinol binding site of cytochrome b 6 . The role of the Rieske protein in forward and backward electron transfer reactions is discussed.

Control of the nitric oxide-cytochrome c oxidase signaling pathway under pathological and physiological conditions.

Prominent among the mechanisms of interaction of nitric oxide (NO) with intracellular targets are the reactions with heme proteins. For example, the mechanism through which NO induces synthesis of the second messenger cyclic GMP involves the binding of NO to the heme in soluble guanylate cyclase. It has only recently been appreciated that NO binding to the binuclear oxygen binding site in cytochrome c oxidase may also serve as a signal transduction pathway. We postulate that NO is uniquely positioned to control mitochondrial respiration and in doing so regulates oxygen gradients within the cell. In this short overview the mechanisms of NO-dependent regulation of mitochondrial function will be discussed in the context of some of the biological and physiological consequences.

Identification of the residues involved in stabilization of the semiquinone radical in the high-affinity ubiquinone binding site in cytochrome bo(3) from Escherichia coli by site-directed mutagenesis and EPR spectroscopy.

During turnover of cytochrome bo(3) from Escherichia coli, a semiquinone radical is stabilized in a high-affinity binding site. To identify binding partners of this radical, site-directed mutants have been designed on the basis of a recently modeled quinone binding site (Abramson et al., 2000). The R71H, H98F, D75H, and I102W mutant enzymes were found to show very little or no quinol oxidase activity. The thermodynamic and EPR spectroscopic properties of semiquinone radicals in these mutants were characterized. For the H98F and the R71H mutants, no EPR signal of the semiquinone radical was observed in the redox potential range from -100 to 250 mV. During potentiometric titration of the D75H mutant enzyme, a semiquinone signal was detected in the same potential range as that of the wild-type enzyme. However, the EPR spectrum of the D75H mutant lacks the characteristic hyperfine structure of the semiquinone radical signal observed in the wild-type oxidase, indicating that D75 or the introduced His, interacts with the semiquinone radical. For the I102W mutant, a free radical signal was observed with a redox midpoint potential downshifted by about 200 mV. On the basis of these observations, it is suggested that R71, D75, and H98 residues are involved in the stabilization of the semiquinone state in the high-affinity binding site. Details of the possible binding motif and mechanistic implications are discussed.

Patterning and Immobilization of Cytochrome C Using Conducting Sulfonated Polyaniline Micro Arrays

A cytochrome c onto photochemically patterned conducting sulfonated polyaniline (SPAN) was immobilized by using a self-assembly technique. The SPAN polymer was employed as a specific binding site for cytochrome c. The electrical conductivity of SPAN was allowed by doping of HCl. Physical and electrochemical properties of the self-assembled cytochrome c monolayer were investigated from the measurements of cyclic voltammetry and atomic force microscopy (AFM). Especially, the electrochemical activity of self-assembled cytochrome c immobilized on the SPAN with a high electrical conductivity was superior.

Patterning and Immobilization of Cytochrome C Using Conducting Sulfonated Polyaniline Micro Arrays

A cytochrome c onto photochemically patterned conducting sulfonated polyaniline (SPAN) was immobilized by using a self-assembly technique. The SPAN polymer was employed as a specific binding site for cytochrome c. The electrical conductivity of SPAN was allowed by doping of HCl. Physical and electrochemical properties of the self-assembled cytochrome c monolayer were investigated from the measurements of cyclic voltammetry and atomic force microscopy (AFM). Especially, the electrochemical activity of self-assembled cytochrome c immobilized on the SPAN with a high electrical conductivity was superior.

Electron Transfer Binding Site of Cytochrome c to Carboxylic Acid-Terminated Alkanethiol Self-assembled Monolayers Immobilized on Gold Electrode*

It is proposed that Lys-13 of mammalian cytochrome cfacilitates the most efficient electron transfer (ET) pathway to the carboxylate terminus of alkanethiol self-assembled monolayers (SAM) on gold electrodes. In order to confirm the proposed ET pathway, the ET reaction rate of a rat cytochrome c mutant (RC9K13A), in which lysine-13 is replaced by alanine, is measured at a 3-mercaptopropionate SAM on a gold electrode. The ET rate of K13A is more than six orders of magnitude smaller than that of the native one. In the mutant, Ala-13 can no longer facilitate the ET pathway. Based on the measurements, the potential candidate for the binding site of RC9K13A is Lys-8.