Respiratory cytochrome c oxidase can be efficiently reduced by the photosynthetic redox proteins cytochrome c6 and plastocyanin in cyanobacteria

Plastocyanin and cytochrome c6 are two small soluble electron carriers located in the intrathylacoidal space of cyanobacteria. Although their role as electron shuttle between the cytochrome b6f and photosystem I complexes in the photosynthetic pathway is well established, their participation in the respiratory electron transport chain as donors to the terminal oxidase is still under debate. Here, we present the first time-resolved analysis showing that both cytochrome c6 and plastocyanin can be efficiently oxidized by the aa3 type cytochrome c oxidase in Nostoc sp. PCC 7119. The apparent electron transfer rate constants are ca. 250 and 300 s(-1) for cytochrome c6 and plastocyanin, respectively. These constants are 10 times higher than those obtained for the oxidation of horse cytochrome c by the oxidase, in spite of being a reaction thermodynamically more favourable.     

Computation of threshold conditions for epidemiological models and global stability of the disease-free equilibrium (DFE)

One goal of this paper is to give an algorithm for computing a threshold condition for epidemiological systems arising from compartmental deterministic modeling. We calculate a threshold condition T(0) of the parameters of the system such that if T(0)<1 the disease-free equilibrium (DFE) is locally asymptotically stable (LAS), and if T(0)>1, the DFE is unstable. The second objective, by adding some reasonable assumptions, is to give, depending on the model, necessary and sufficient conditions for global asymptotic stability (GAS) of the DFE. In many cases, we can prove that a necessary and sufficient condition for the global asymptotic stability of the DFE is R(0)< or =1, where R(0) is the basic reproduction number [O. Diekmann, J.A. Heesterbeek, Mathematical Epidemiology of Infectious Diseases: Model Building, Analysis and Interpretation, Wiley, New York, 2000]. To illustrate our results, we apply our techniques to examples taken from the literature. In these examples we improve the results already obtained for the GAS of the DFE. We show that our algorithm is relevant for high dimensional epidemiological models.     

Transmembrane proton translocation by cytochrome c oxidase

Respiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O₂-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O₂ reduction and how these reactions might be energetically coupled to proton pumping across the membrane.     

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H₂O₂ generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.     

Reaction of wild-type and Glu243Asp variant yeast cytochrome c oxidase with O2

We have studied internal electron transfer during the reaction of Saccharomyces cerevisiae mitochondrial cytochrome c oxidase with dioxygen. Similar absorbance changes were observed with this yeast oxidase as with the previously studied Rhodobacter sphaeroides and bovine mitochondrial oxidases, which suggests that the reaction proceeds along the same trajectory. However, notable differences were observed in rates and electron-transfer equilibrium constants of specific reaction steps, for example the ferryl (F) to oxidized (O) reaction was faster with the yeast (0.4 ms) than with the bovine oxidase (~ 1 ms) and a larger fraction Cu_A was oxidized with the yeast than with the bovine oxidase in the peroxy (P_R) to F reaction. Furthermore, upon replacement of Glu243, located at the end of the so-called D proton pathway, by Asp the P_R → F and F → O reactions were slowed by factors of ~ 3 and ~ 10, respectively, and electron transfer from Cu_A to heme a during the P_R → F reaction was not observed. These data indicate that during reduction of dioxygen protons are transferred through the D pathway, via Glu243, to the catalytic site in the yeast mitochondrial oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Crystal structure of oxidized cytochrome c(6A) from Arabidopsis thaliana

Compared with algal and cyanobacterial cytochrome c(6), cytochrome c(6A) from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c(6A) from Arabidopsis thaliana at 1.5 Angstrom resolution in order to help elucidate its function. The overall structure of cytochrome c(6A) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c(6A). Our model provides a chemical basis for the known low redox potential of cytochrome c(6A) which makes it an unsuitable electron carrier between cytochrome b(6)f and PSI.     

Nitric oxide, cytochrome c oxidase and myoglobin: competition and reaction pathways

It is relevant to cell physiology that nitric oxide (NO) reacts with both cytochrome oxidase (CcOX) and oxygenated myoglobin (MbO(2)). In this respect, it has been proposed [Pearce, L.L., et al. (2002) J. Biol. Chem. 277, 13556-13562] that (i) CcOX in turnover out-competes MbO(2) for NO, and (ii) NO bound to reduced CcOX is "metabolized" in the active site to nitrite by reacting with O(2). In contrast, rapid kinetics experiments reported in this study show that (i) upon mixing NO with MbO(2) and CcOX in turnover, MbO(2) out-competes the oxidase for NO and (ii) after mixing nitrosylated CcOX with O(2) in the presence of MbO(2), NO (and not nitrite) dissociates from the enzyme causing myoglobin oxidation.     

Identification of an actin-binding site in p47phox an organizer protein of NADPH oxidase

Actin has been reported to enhance the superoxide-generating activity of neutrophil NADPH oxidase in a cell-free system and to interact with p47phox, a regulatory subunit of the oxidase. In the present study, we searched for an actin-binding site in p47phox by far-western blotting and blot-binding assays using truncated forms of p47phox. The amino-acid sequence 319-337 was identified as an actin-binding site, and a synthetic peptide of this sequence bound to actin. The sequence shows no homology to other known actin-binding motifs. It is located in the autoinhibitory region of p47phox and includes Ser-328, a phosphorylation site essential for unmasking. Although a phosphorylation-mimetic p47phox mutant bound to actin with a lower affinity than the wild type, the same mutant interacted with filamentous actin more efficiently than the wild type. A mutant peptide p47phox (319-337, Ser328Glu) bound to filamentous actin more tightly than to monomer actin. These results suggest that p47phox moves to cortical actin when it becomes unmasked in the cells.     

Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is shown to be dependent on a new assembly factor designated Coa1 that associates with the mitochondrial inner membrane. Translation of the mitochondrial-encoded subunits of CcO occurs normally in coa1Delta cells, but these subunits fail to accumulate. The respiratory defect in coa1Delta cells is suppressed by high-copy MSS51, MDJ1 and COX10. Mss51 functions in Cox1 translation and elongation, whereas Cox10 participates in the biosynthesis of heme a, a key cofactor of CcO. Respiration in coa1Delta and shy1Delta cells is enhanced when Mss51 and Cox10 are coexpressed. Shy1 has been implicated in formation of the heme a3-Cu(B) site in Cox1. The interaction between Coa1 and Cox1, and the physical and genetic interactions between Coa1 and Mss51, Shy1 and Cox14 suggest that Coa1 coordinates the transition of newly synthesized Cox1 from the Mss51:Cox14 complex to the heme a cofactor insertion involving Shy1. coa1Delta cells also display a mitochondrial copper defect suggesting that Coa1 may have a direct link to copper metallation of CcO.     

Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.     

Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH

Background

In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O₂ is linked to proton uptake from solution to form H₂O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.

Methods

In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O₂. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.

Results

The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O₂ reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.

Conclusions

The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.

General significance

These results provide new insights into the function of cytochrome c oxidase.     

Mechanism and energetics by which glutamic acid 242 prevents leaks in cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of aerobic respiration. The energy released from the reduction of molecular oxygen to water is used to pump protons across the mitochondrial or bacterial membrane. The pump function introduces a mechanistic requirement of a valve that prevents protons from flowing backwards during the process. It was recently found that Glu-242, a key amino acid in transferring protons to be pumped across the membrane and to the site of oxygen reduction, fulfils the function of such a valve by preventing simultaneous contact to the pump site and to the proton-conducting D-channel (Kaila V.R.I. et al. Proc. Natl. Acad. Sci. USA 105, 2008). Here we have incorporated the valve model into the framework of the reaction mechanism. The function of the Glu valve is studied by exploring how the redox state of the surrounding metal centers, dielectric effects, and membrane potential, affects the energetics and leaks of this valve. Parallels are drawn between the dynamics of Glu-242 and the long-standing obscure difference between the metastable O_H and stable O states of the binuclear center. Our model provides a suggestion for why reduction of the former state is coupled to proton translocation while reduction of the latter is not.     

Perturbation avalanches and criticality in gene regulatory networks

Boolean networks are simplified models of gene regulatory networks. We derive an approximation of the size distribution of perturbation avalanches in Boolean networks based on known results in the theory of branching processes. We show numerically that the approximation works well for different kinds of Boolean networks. It has been suggested that gene regulatory networks may be dynamically critical. To study this, as an application of the presented theory we present a novel method for estimating an order parameter from microarray data. According to the available data and our method, we find that gene regulatory networks appear to be stable and reside near the phase transition between order and chaos.     

The antimicrobial peptide parabutoporin competes with p47(phox) as a PKC-substrate and inhibits NADPH oxidase in human neutrophils

We investigated parabutoporin (PP), an antimicrobial scorpion peptide, to understand its inhibition on NADPH oxidase in human PMN. We show that PP is a good substrate for all PKC-isotypes, implicated in the activation of NADPH oxidase, and acts as a potent competitive inhibitor of in vitro p47(phox)-phosphorylation by PKC-alpha, -betaI, -betaII and -delta, but not PKC-zeta. In PMN, PP also inhibits the PMA-stimulated phosphorylation of p47(phox) and its subsequent translocation. In contrast, PP affects the PKC-independent activation to a much lesser degree. This indicates that PP inhibits the activation of NADPH oxidase at submicromolar concentrations in a strongly PKC-dependent manner.     

Testing polyols' compatibility with Gibbs energy of stabilization of proteins under conditions in which they behave as compatible osmolytes

It is generally believed that compatible osmolytes stabilize proteins by shifting the denaturation equilibrium, native state <--> denatured state toward the left. We show here that if osmolytes are compatible with the functional activity of the protein at a given pH and temperature, they should not significantly perturb this denaturation equilibrium under the same experimental conditions. This conclusion was reached from the measurements of the activity parameters (K(m) and k(cat)) and guanidinium chloride-induced denaturations of lysozyme and ribonuclease-A in the presence of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at pH 7.0 and 25 degrees C.     

The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1

When the eicosapentaenoic acid (EPA)-deficient mutant strain IK-1Delta8 of the marine EPA-producing Shewanella marinintestina IK-1 was treated with various concentrations of hydrogen peroxide (H(2)O(2)), its colony-forming ability decreased more than that of the wild type. Protein carbonylation, induced by treating cells with 0.01 mM H(2)O(2) under bacteriostatic conditions, was enhanced only in cells lacking EPA. The amount of cells recovered from the cultures was decreased more significantly by the presence of H(2)O(2) for cells lacking EPA than for those producing EPA. Treatment of the cells with 0.1 mM H(2)O(2) resulted in much lower intracellular concentrations of H(2)O(2) being consistently detected in cells with EPA than in those without EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2) in S. marinintestina IK-1.     

Thermodynamic and EPR studies of slowly relaxing ubisemiquinone species in the isolated bovine heart complex I

Previously, we investigated ubisemiquinone (SQ) EPR spectra associated with NADH-ubiquinone oxidoreductase (complex I) in the tightly coupled bovine heart submitochondrial particles (SMP). Based upon their widely differing spin relaxation rate, we distinguished SQ spectra arising from three distinct SQ species, namely SQ(Nf) (fast), SQ(Ns) (slow), and SQ(Nx) (very slow). The SQ(Nf) signal was observed only in the presence of the proton electrochemical gradient (deltamu(H)(+)), while SQ(Ns) and SQ(Nx) species did not require the presence of deltamu(H+). We have now succeeded in characterizing the redox and EPR properties of SQ species in the isolated bovine heart complex I. The potentiometric redox titration of the g(z,y,x)=2.00 semiquinone signal gave the redox midpoint potential (E(m)) at pH 7.8 for the first electron transfer step [E(m1)(Q/SQ)] of -45 mV and the second step [E(m2)(SQ/QH(2))] of -63 mV. It can also be expressed as [E(m)(Q/QH(2))] of -54 mV for the overall two electron transfer with a stability constant (K(stab)) of the SQ form as 2.0. These characteristics revealed the existence of a thermodynamically stable intermediate redox state, which allows this protein-associated quinone to function as a converter between n=1 and n=2 electron transfer steps. The EPR spectrum of the SQ species in complex I exhibits a Gaussian-type spectrum with the peak-to-peak line width of approximately 6.1 G at the sample temperature of 173 K. This indicates that the SQ species is in an anionic Q(-) state in the physiological pH range. The spin relaxation rate of the SQ species in isolated complex I is much slower than the SQ counterparts in the complex I in situ in SMP. We tentatively assigned slow relaxing anionic SQ species as SQ(Ns), based on the monophasic power saturation profile and several fold increase of its spin relaxation rate in the presence of reduced cluster N2. The current study also suggests that the very slowly relaxing SQ(Nx) species may not be an intrinsic complex I component. The functional role of SQ(Ns) is further discussed in connection with the SQ(Nf) species defined in SMP in situ.