Protein Surface Mimetics: Understanding How Ruthenium Tris(Bipyridines) Interact with Proteins.

Protein surface mimetics achieve high-affinity binding by exploiting a scaffold to project binding groups over a large area of solvent-exposed protein surface to make multiple cooperative noncovalent interactions. Such recognition is a prerequisite for competitive/orthosteric inhibition of protein-protein interactions (PPIs). This paper describes biophysical and structural studies on ruthenium(II) tris(bipyridine) surface mimetics that recognize cytochrome (cyt) c and inhibit the cyt c/cyt c peroxidase (CCP) PPI. Binding is electrostatically driven, with enhanced affinity achieved through enthalpic contributions thought to arise from the ability of the surface mimetics to make a greater number of noncovalent interactions than CCP with surface-exposed basic residues on cyt c. High-field natural abundance 1 H,15 N HSQC NMR experiments are consistent with surface mimetics binding to cyt c in similar manner to CCP. This provides a framework for understanding recognition of proteins by supramolecular receptors and informing the design of ligands superior to the protein partners upon which they are inspired.

Subclinical carbon monoxide limits apoptosis in the developing brain after isoflurane exposure.

BACKGROUND: Volatile anesthetics cause widespread apoptosis in the developing brain. Carbon monoxide (CO) has antiapoptotic properties, and exhaled endogenous CO is commonly rebreathed during low-flow anesthesia in infants and children, resulting in subclinical CO exposure. Thus, we aimed to determine whether CO could limit isoflurane-induced apoptosis in the developing brain. METHODS: Seven-day-old male CD-1 mouse pups underwent 1-hour exposure to 0 (air), 5, or 100 ppm CO in air with or without isoflurane (2%). We assessed carboxyhemoglobin levels, cytochrome c peroxidase activity, and cytochrome c release from forebrain mitochondria after exposure and quantified the number of activated caspase-3 positive cells and TUNEL positive nuclei in neocortex, hippocampus, and hypothalamus/thalamus. RESULTS: Carboxyhemoglobin levels approximated those expected in humans after a similar time-weighted CO exposure. Isoflurane significantly increased cytochrome c peroxidase activity, cytochrome c release, the number of activated caspase-3 cells, and TUNEL positive nuclei in the forebrain of air-exposed mice. CO, however, abrogated isoflurane-induced cytochrome c peroxidase activation and cytochrome c release from forebrain mitochondria and decreased the number of activated caspase-3 positive cells and TUNEL positive nuclei after simultaneous exposure with isoflurane. CONCLUSIONS: Taken together, the data indicate that CO can limit apoptosis after isoflurane exposure via inhibition of cytochrome c peroxidase depending on concentration. Although it is unknown whether CO directly inhibited isoflurane-induced apoptosis, it is possible that low-flow anesthesia designed to target rebreathing of specific concentrations of CO may be a desired strategy to develop in the future in an effort to prevent anesthesia-induced neurotoxicity in infants and children.

A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death.

The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of haemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids that blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its haem-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 h before through 24 h after the irradiation.

beta-Secretase expression in normal and functionally deprived rat olfactory bulbs: inverse correlation with oxidative metabolic activity.

Cerebral hypometabolism, mitochondrial dysfunction, and beta-amyloid peptide (Abeta) accumulation are well-characterized manifestations of Alzheimer's disease (AD). beta-Secretase (BACE) is a prerequisite for amyloidogenesis, and it is up-regulated in sporadic AD. To explore a potential in vivo mechanism by which Abeta production is modulated by neuronal activity and/or oxidative metabolism, we compared BACE expression with cytochrome c oxidase (CO) or succinic dehydrogenase (SDH) activity in normal and functionally deprived adult rat olfactory bulb. In normal bulb, BACE was expressed predominantly in the glomerular layer, but labeling intensity within individual glomeruli varied substantially. A strong negative correlation existed between BACE labeling intensity and CO or SDH activity among individual glomeruli. Unilateral naris occlusion resulted in elevated glomerular BACE labeling in the deprived bulbs relative to the nondeprived counterparts, which was correlated with decreased CO activity in the same anatomic location. Enhanced BACE labeling was confirmed by measurements of elevated protein levels, enzymatic activity, and beta-site cleavage products of amyloid precursor protein in bulb extracts. Our findings reveal a negative regulation of BACE expression by physiological neuronal activity and an intrinsic inverse correlation between BACE expression and oxidative metabolism at the first synapse on the olfactory pathway. The results point to a biological role of BACE in synapse function and plasticity as well as a potential mechanism whereby reduced neuronal activity or metabolism could lead to amyloid overproduction in synaptic terminals.

Protonation and inhibition of Nitrosomonas europaea cytochrome c peroxidase observed with protein film voltammetry.

The impact of protonation and inhibitor binding of the diheme cytochrome c peroxidase (CCP) from Nitrosomonas europaea has been examined by the technique of catalytic protein film voltammetry (PFV). Previous efforts have shown that the low-potential heme active site (L) binds substrate and yields electrocatalysis at an pyrolytic graphite edge electrode, with properties evocative of a high-potential intermediate, with E(m)>540mV (vs. normal hydrogen electrode) [A.L. Bradley, S.E. Chobot, D.M. Arciero, A.B. Hooper, S. J. Elliott, J. Biol. Chem. 279 (2004) 13297-13300]. Here we demonstrate through similar experiments that catalytic PFV generates limiting currents which allow for electrochemically-detected enzymology of the Ne CCP: such as the demonstration that pH-dependent Michaelis-Menten constants (K(m) values) reveal a pK(a) value of 6.5 associated with the "ES" complex. Further, the direct electrocatalysis is shown in the presence of known inhibitors (cyanide and azide), indicating that inhibitor binding occurs at L, and shifts the resulting catalytic midpoint potential in a negative direction. Michaelis-Menten treatment of the limiting currents generated in the presence of variable concentrations of inhibitors showed that cyanide behaved as a competitive inhibitor with a K(i) value of 0.15muM; azide revealed a mixed-mode of inhibition. The observed data were found to support a previous model of electrocatalysis, and the role of proton transfer chemistry in the active site is discussed in terms of a structural model.

Disruption of protein-protein interactions using nanoparticles: inhibition of cytochrome c peroxidase.

Functionalized gold nanoparticles bind selectively to cytochrome c or cytochrome c peroxidase and inhibit enzyme turnover.

Disruption of protein-protein interactions: design of a synthetic receptor that blocks the binding of cytochrome c to cytochrome c peroxidase.

Synthetic receptor 1 has been found via fluorescence titration to compete effectively with cytochrome c peroxidase for binding cytochrome c (Cc), forming 1:1 Cc:1 complex with a binding constant of (3 +/- 1) x 10(8) M-1, and to disrupt Cc: Apaf-1 complex, a key adduct in apoptosis.

Cytochrome c peroxidase from Methylococcus capsulatus Bath.

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively.

Small substrates and cytochrome c are oxidized at different sites of cytochrome c peroxidase.

Modeling studies suggest that electrons are transferred from cytochrome c to cytochrome c peroxidase (CcP) with cytochrome c predominantly bound at a site facing the gamma-meso edge of the CcP prosthetic heme group (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330). As shown here, guaiacol and ferrocyanide are oxidized at a different site of CcP. Thus, the oxidations of cytochrome c and guaiacol are differentially inactivated by phenylhydrazine and sodium azide. The loss of guaiacol oxidation activity correlates with covalent binding of 1 equivalent of [14C]phenylhydrazine to the protein, whereas the slower loss of cytochrome c activity correlates with the appearance of a 428-nm absorbance maximum attributed to the formation of a sigma-phenyl-iron heme complex. The delta-meso-phenyl and 8-hydroxymethyl derivatives of heme are formed as minor products. Catalytic oxidation of azide to the azidyl radical results in inactivation of CcP and formation of delta-meso-azidoheme. Reconstitution of apo-CcP with delta-meso-azido-, -ethyl-, and -(2-phenylethyl)heme yields holoproteins that give compound I species with H2O2 and exhibit 80, 59, and 31%, respectively, of the control kcat value for cytochrome c oxidation but little or no guaiacol or ferrocyanide oxidizing activity. Conversely, CcP reconstituted with gamma-meso-ethylheme is fully active in the oxidation of guaiacol and ferrocyanide but only retains 27% of the cytochrome c oxidizing activity. These results indicate that guaiacol and ferrocyanide are primarily oxidized near the delta-meso-heme edge rather than, like cytochrome c, at a surface site facing the gamma-meso edge.

Crystal structure of nitric oxide inhibited cytochrome c peroxidase.

We have collected X-ray diffraction data from a crystal of cytochrome c peroxidase (CCP) complexed with the inhibitor nitric oxide to a resolution of 2.55 A. A difference Fourier map shows density indicating the NO ligand is bound to the heme iron at the sixth coordination site in a bent configuration. Structural adjustments were determined by least-squares refinement that yielded an agreement residual of R = 0.18. The orientation of the ligand, tilting toward Arg-48, causes adjustment in the position of this nearby polar side chain. As a model for the substrate hydrogen peroxide, this geometry is consistent with the suggestion that Arg-48 serves to polarize the O-O peroxide bond to promote heterolytic cleavage of the bond [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205]. Strong difference density is also observed near residues 190-194, especially around the indole ring of Trp-191. The density indicates movement of the indole ring away from the proximal His-175 imidazole ring by about 0.25 A, which appears to cause perturbation of the neighboring residues. The response of Trp-191 on the proximal side of the heme to binding nitric oxide on the distal side probably results from delocalization of the electron density of the ligand. Relevant to this is the recent finding that a mutant in which Trp-191 is replaced by phenylalanine has dramatically reduced activity, less than 0.05% of the parent activity [Mauro, J. M., Fishel, L. A., Hazzard, J. T., Meyer, T. E., Tollin, G., Cusanovich, M. A., & Kraut, J. (1988) Biochemistry 27, 6243-6256].(ABSTRACT TRUNCATED AT 250 WORDS)

The crystal structure of fluoride-inhibited cytochrome c peroxidase.

The three-dimensional crystal structure of yeast cytochrome c peroxidase complexed with fluoride (F- or HF) has been determined by difference Fourier techniques and partially refined at 2.5-A resolution. Fluoride binding induces significant perturbations of the enzyme structure of the distal side of the heme. The major effect occurs at the active-site arginine residue (Arg-48) which moves about 2 A in order to optimize hydrogen-bonded interactions with the fluorine atom. A small readjustment of the distal histidine (His-52), about 0.5 A, is also seen upon fluoride binding. Additionally, a hydrogen-bonded network of 4 water molecules at the active site is reorganized. No significant movements are detectable in either the heme itself or in the proximal histidine ligand. These observations imply that movement of the Arg-48 side chain may play a key role in the enzymic mechanism of cytochrome c peroxidase. Although we cannot unequivocally determine whether fluoride is bound as HF or F-, the hydrogen-bonding pattern around the ligand points to the protonated form. Structural comparison suggests that there is a difference between the tautomeric state of the imidazole side chain of the distal histidine in cytochrome c peroxidase and of the similarly positioned distal histidine in the globins. This difference accounts for the observation that cytochrome c peroxidase preferentially binds the protonated form of ligands, whereas the globins bind the anionic form. The tautomer indicated by the peroxidase structure is the one required for acid base catalysis (Poulos, T.L., and Finzel, B. C. (1984) in Peptide and Protein Reviews (Decker, M., ed) in press).

The cytochrome c peroxidase activity of cytochrome oxidase.

I have found that mammalian cytochrome oxidase catalyzes the peroxidatic oxidation of ferrocytochrome c under strictly anaerobic conditions. An apparent Km value for ferrocytochrome c was 2 microM, and a second order rate constant, estimated as an extrapolated value, was 1.4 X 10(6) M-1 s-1 at pH 7.4 at 25 degrees C. These values were quite similar to the corresponding values of 6.4 microM and 1.9 X 10(6) M-1 s-1 determined for the intrinsic oxidase activity. The rate of the peroxidatic oxidation showed a hyperbolic dependence on the concentration of hydrogen peroxide, and the apparent Km value ws 0.18 mM. Cyanide and azide at 0.1 mM inhibited the peroxidase activity by 100 and 98%, respectively, whereas, under carbon monoxide at 750 mm Hg, 10% of the activity still remained. Under air, cytochrome oxidase acted simultaneously as oxidase and peroxidase.

The effects of miconazole on the ultrastructure of Candida albicans.

Ultrastructural changes in Candida albicans induced by increasing concentrations of miconazole in vitro are described. Fungistatic concentrations (10(-8) to 10(-7) M) induced minimal morphologic changes at the cell periphery. At 10(-6) M cell volume increased and peroxisomes became numerous in the cell interior. The minimal fungicidal dose of 10(-5) M caused severe damage to most of the cell population and a total fungicidal dose of 10(-4) M caused total internal cellular necrosis even when cell walls remained intact. It is suggested that miconazole inhibits the peroxidative enzymes cytochrome c-peroxidase and catalase. Cell necrosis then results from peroxide accumulation.

Pseudomonas cytochrome c peroxidase. XII. Product inhibition studies.

Kinetic studies of the reaction mechanism of Pseudomonas cytochrome c peroxidase (PaCCP) were made by the method of product inhibition using oxidized cytochrome C (551 P.aeruginosa) and oxidized Pseudomonas azurin as products. Inhibition by the two oxidized substrates was linearly non-competitive towards the respective reduced electron donor and towards hydrogen peroxide. Although a full kinetic analysis is experimentally impossible in a peroxidase-type reaction, the results do provide some evidence in favour of an ordered reaction mechanism in which hydrogen peroxide is the first to add to PaCCP and electron donor the second.