Reactivity characteristics of cytochrome c, cytochrome oxidase and the electrostatic cytochrome c - cytochrome oxidase complex /

The mechanism whereby cytochrome £ oxidase catalyses elec-. tron transfer from cytochrome £ to oxygen remains an unsolved problem. Polarographic and spectrophotometric activity measurements of purified, particulate and soluble forms of beef heart mitochondrial cytochrome c oxidase presented in this thesis confirm the following characteristics of the steady-state kinetics with respect to cytochrome £: (1) oxidation of ferrocytochrome c is first order under all conditions. -(2) The relationship between sustrate concentration and velocity is of the Michaelis-Menten type over a limited range of substrate. concentrations at high ionic strength. (3) ~he reaction rate is independent from oxygen concentration until very low levels of oxygen. (4) "Biphasic" kinetic plots of enzyme activity as a function of substrate concentration are found when the range of cytochrome c concentrations is extended; the biphasicity ~ is more apparent in low ionic strength buffer. These results imply two binding sites for cytochrome £ on the oxidase; one of high affinity and one of low affinity with Km values of 1.0 pM and 3.0 pM, respectively, under low ionic strength conditions. (5) Inhibition of the enzymic rate by azide is non-c~mpetitive with respect to cytochrome £ under all conditions indicating an internal electron transfer step, and not binding or dissociation of £ from the enzyme is rate limiting. The "tight" binding of cytochrome '£ to cytochrome c oxidase is confirmed in column chromatographic experiments. The complex has a cytochrome £:oxidase ratio of 1.0 and is dissociated in media of high ionic strength. Stopped-flow spectrophotometric studies of the reduction of equimolar mixtures and complexes of cytochrome c and the oxidase were initiated in an attempt to assess the functional relevance of such a complex. Two alternative routes -for reduction of the oxidase, under conditions where the predominant species is the £ - aa3 complex, are postulated; (i) electron transfer via tightly bound cytochrome £, (ii) electron transfer via a small population of free cytochrome c interacting at the "loose" binding site implied from kinetic studies. It is impossible to conclude, based on the results obtained, which path is responsible for the reduction of cytochrome a. The rate of reduction by various reductants of free cytochrome £ in high and low ionic strength and of cytochrome £ electrostatically bound to cytochrome oxidase was investigated. Ascorbate, a negatively charged reagent, reduces free cytochrome £ with a rate constant dependent on ionic strength, whereas neutral reagents TMPD and DAD were relatively unaffected by ionic strength in their reduction of cytochrome c. The zwitterion cysteine behaved similarly to uncharged reductants DAD and TI~PD in exhibiting only a marginal response to ionic strength. Ascorbate reduces bound cytochrome £ only slowly, but DAD and TMPD reduce bound cytochrome £ rapidly. Reduction of cytochrome £ by DAD and TMPD in the £ - aa3 complex was enhanced lO-fold over DAD reduction of free £ and 4-fold over TMPD reduction of free c. Thus, the importance of ionic strength on the reactivity of cytochrome £ was observed with the general conclusion being that on the cytochrome £ molecule areas for anion (ie. phosphate) binding, ascorbate reduction and complexation to the oxidase overlap. The increased reducibility for bound cytochrome £ by reductants DAD and TMPD supports a suggested conformational change of electrostatically bound c compare.d to free .£. In addition, analysis of electron distribution between cytochromes £ and a in the complex suggest that the midpotential of cytochrome ~ changes with the redox state of the oxidase. Such evidence supports models of the oxidase which suggest interactions within the enzyme (or c - enzyme complex) result in altered midpoint potentials of the redox centers.

Effect of citrate on the redox properties of cytochrome C and its kinetic and binding behaviour with cytochrome oxidase /

Increasing citrate concentration, at constant ionic strength (30 mM) decreases the rate of cytochrome ~ reduction by ascorbate. This effect is also seen at both high (600 mM) and low (19 mM) ionic strengths, and the Kapp for citrate increases with increasing ionic strength. Citrate binds d both ferri -and ferrocytochrome ~, but with a lower affinity for the latter form (Kox . .red d = 2 mM, Kd = 8 mM) as shown by an equilibrium assay with N,N,N',N', Tetramethyl E- phenylenediamine. The reaction of ferricytochrome ~with cyanide is also altered in the presence of citrate: citrate increases the K~PP for cyanide. Column chromatography of cytochrome ~-cytochrome oxidase mixtures shows citrate increases the dissociation constant of the complex. These results are confirmed in kinetic assays for the "loose"site (Km = 20 pM) only. The effect of increasing citrate observable at the "tight" site (Km = 0.25 pM) is on the turnover number and not on the K . These results suggest a mechanism m where anion binding to cytochrome £ at the tight site affects the equilibrium between two forms of cytochrome c bound cytochrome oxidase: an active and an inactive one.

Analysis of a temperature sensitive mutation affecting aldehyde oxidase activity in Drosophila melanogaster

A strain of Drosophila melanogaster (mid america stock culture no. hl16) has been reported to be deficient in aldehyde oxidase activity (Hickey and Singh 1982). This strain was characterized during the course of this study and compared to other mutant strains known to be deficient in aldehyde oxidase activity. During the course of this investigation, the hl16 strain was found to be temperature sensitive in its viability. It was found that the two phenotypes, the enzyme deficiency, and the temperature sensitive lethality were the result of two different mutations, both mapping to the X-chromosome. These two mutations were found to be separable by recombination. The enzyme deficiency was found to map to the same locus as the cinnamon mutation, another mutation which affects aldehyde oxidase production. The developmental profile of aldehyde oxidase in the hl16 strain was compared to the developmental profile in the Canton S wild type strain. The aldehyde oxidase activity in adult hl16 individuals was also compared to that of various other strains. It was also found that the aldehyde oxidase activity was temperature sensitive in the adult flies. The temperature sensitive lethality mutation was mapped to position 1-0.1.

Comparative study of bovine heart and bacillus subtilis cytochrome c oxidase vesicles and the influence of bulk pH on cytochrome c oxidase components

Studies on the steady state behavior of soluble cytochrome c oxidase are extensive. These studies have examined the influence of ionic strength and pH and may provide answers to questions such as the link between proton translocation and charge separation. The present study examined the influence of external bulk pH on ApH formation, biphasic kinetics, and steady state reduction of cytochromes c and a of cytochrome c oxidase in proteoliposomes. Bulk pH has an appreciable effect on ApH formation and steady state reduction levels of cytochromes c and 8. Bulk pH affected total Vmax and Km at the low affinity binding site of cytochrome c. This study also examined the influence of bovine serum albumin and free fatty acids on proton pumping activity in bovine heart proteoliposomes. Proton pumping activity decreased after treatment with BSA, and was subsequently reinstated after further treatment with FFA. Much study in the superfamily of haem/copper oxidases has recently been devoted to the bacterial oxidases. The present study has examined some protein composition characteristics and bioenergetic features of Bacillus subtilis cytochrome caa3 oxidase. Results provide evidence for the structural composition of the enzyme in relation to the covalently bound cytochrome c to the oxidas~. Bioenergetically, caa3 COV showed appreciable proton pumping activity. Steady state analysis of the caa3 COV showed significantly different cytochrome c and a reduction characteristics compared to the bovine enzyme.

Effects of large anphiphilic ligands upon the spectra and kinetics of cytochrome C oxidase

Cytoch ro me c oxidase (ferrocytochrome c : 02 oxidoreductase ; EC 1.9. 3.1) is the terminal enzyme in the mitochondrial electron transport chain, catalyzing the transfer of electrons from ferrocytochrome c to molecular oxygen. The effects of two large amphiphilic molecules .. valinomycin and dibucaine upon the spectra of the isolated enzyme and upon the activity of both isolated enzyme and enzyme in membrane systems are investigated by using spectrophotometric and oxygen electrode techniques. The results show that both valinomycin and dibucaine change the Soret region of the spectrum and cause a partial inhibition in a concentration range higher than that in which they act as ionophores. It is concluded that both valinomycin and dibucain~ binding induce a conformational change of the protein structure which modifies the spectrum of the a3 CUB centre and diminishes the rate of electron transfer between cytochrome a and the binuclear centre.

Proton translocation by cytochrome c oxidase reconstituted into proteoliposomes

Cytochrome c oxidase .inserted into proteoliposomes translocates protons with a stoichiometry of approx-, imately 0.4-0.6 H+/e- in the presence of valinomycin plus pottasium. The existance .ofsuchproton translocation is .supportedby experiments with lauryl maltoside which abolished the pulses but~~d not inhibit cyt. c binding .or oxidase turnover. Pulses with K3FeCN6 did not induce acidification further supporting vectorial proton transport by cyt ..aa3 . Upon lowering the ionic strength and pulsing with ferrocytochrome c, H+/eratios increased. This increase is attributed to scaler proton release consequent upon cyt.c-phospholipid binding. Oxygen pulses at low ionic strength however did not exhibit this large scaler increase in H+/e- ratios.A-small increase was observed upon .02 pul'sing at·low ionic strengt.h. This increase was KeN and, ,pcep sensitive and thus possibly due to a redox linked scaler deprotonation. Increases in the H+/e- ratio also occurred ifp~lses ,were performed in the presence of nonactin rather.than valinomycin. The fluorescent pH indicator pyranine was internally trapped inaa3 conta~ning "proteoliposomes. Internal alkalinization, as mon,itored by pyranine fluorescence leads to a of approx.imately 0.35 units, which is proportional to electron flux. This internal alkalinization was also DCCD sensitive, being inhibited by approximately 50%. This 50% inhibition of internal alkalinization supports the existance of vectorial proton transport.

Hydrogen sulphide as inhibitor and substrates for the cytochrome c-cytochrome c oxidase system /

It has previously been recognized that the major biochemical toxicity induced by sulphide is due to an inhibition of cytochrome ~ oxidase. Inhibition of this enzyme occurs at 30°C and pH 7.4 with a Ki of approximately 0.2 ~M, and a kon of 104 M-1 s-l, under catalytic conditions. However, the equimo1ar mixture of sulphide and the enzyme shows identical catalytic behaviour to that of the native enzyme. This cannot readily be attributed to rapid dissociation of sulphide, as both spectroscopic and plot analysis indicate the koff value is low. The addition of stoichiometric sulphide to the resting oxidized enzyme gives rise to the appearance of a low-spin ferric-type spectrum not identical with that seen on the addition of excess sulphide to the enzyme aerobically. Sulphide added to the enzyme anaerobically gives rise to another low-spin, probably largely ferric, form which upon admission of oxygen is then converted into a 607 nm species closely resembling Compound C. The 607 nm form is probably the precursor of oxyferricytochrome aa3. The addition of successive a1iquots of Na2S solution to the enzyme induces initial uptake of approximately 3 moles of oxygen per mole of the enzyme. Thus, it is concluded that: 1. the initial product of sulphide-cytochrome c oxidase interaction is not an inhibited form of the enzyme, but the low-spin (oxyferri) ~3+~+ species; 2. a subsequent step in which sulphide reduces cytochrome ~ occurs; 3. the final inhibitory step, in which a further molecule of sulphide binds to the cytochrome ~ iron centre in the cytochrome ~2+~+ species, gives the cytochrome a2+~+-H2S form which is a half-reduced fully inhibited species;4. a 607 run form of the enzyme is produced which may be converted into a catalytically active low-spin (oxyferri) state; and therefore 5. liganded sulphide may be able to reduce the cytochrome 33 -Cu centre without securing the prior reduction of the cytochrome a_ haem group or the Cud centre associated with it.

Effects of large anphiphilic ligands upon the spectra and kinetics of cytochrome C oxidase

Cytoch ro me c oxidase (ferrocytochrome c : 02 oxidoreductase ; EC 1.9. 3.1) is the terminal enzyme in the mitochondrial electron transport chain, catalyzing the transfer of electrons from ferrocytochrome c to molecular oxygen. The effects of two large amphiphilic molecules - valinomycin and dibucaine upon the spectra of the isolated enzyme and upon the activity of both isolated enzyme and enzyme in membrane systems are investigated by using spectrophotometric and oxygen electrode techniques. The results show that both valinomycin and dibucaine change the Soret region of the speetrum and cause a partial inhibition in a concentration range higher than that in which they act as ionophores. It is concluded that both valinomycin and dibucaine binding induce a conformational change of the protein structure which modifies the spectrum of the a3 CUB centre and diminishes the rate of electron transfer between cytochrome a and the binuclear centre.

Expression and function of endothelin and its receptors in vascular adventitial fibroblasts

Objective: The adventitia has been recognized to play important roles in vascular oxidative stress, remodelling and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin-1 (ET-1) in response to angiotensin II (ANG II). However, the mechanisms by which ANG II induces ET-1 expression are unknown. It is also unclear whether the ET-1 receptors are expressed in the adventitia. We therefore examined the role of oxidative stress in the regulation of ET-1. We also investigated the expression of both the ETA and ETB receptors and the roles of these two types of receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Methods and Results: Adventitial fibroblasts were isolated and cultured from the thoracic mouse aorta. Cells were treated with ANG II (lOOnM), ET-1 (lOpM), NADPH oxidase inhibitor apocynin (lOOfiM), the superoxide anion scavenger tempol (lOOfiM), the ANG II receptor antagonists (100[aM), losartan (AT| receptor) and PD 1233 19 (AT2 receptor), the ET-1 receptor antagonists (lOOuM), BQ123 (ETA receptor) and BQ788 (ETB receptor), and the ETB receptor agonist (lOOnM) Sarafotoxin 6C. ET-1 peptide levels were determined by ELISA, while ETA ,ETB and collagen levels were determined by Western blot. ANG II increased ET-1 peptide levels in a time-dependent manner reaching significance when incubated for 24 hours. NAD(P)H oxidase inhibitor, apocynin, as well as the superoxide scanverger, tempol, significantly reduced ANG Il-induced ET-1 peptide levels while over-expression of SOD1 (endogenous antioxidant enzyme) significantly decreased ANG Il-induced collagen I expression, therefore implicating reactive oxygen species in the mediation of ET-1. ANG II increased ETA receptor protein as well as collagen in a similar fashion, reaching significance after 4, 6, and 24 hours treatment. ANG II induced collagen was reduced while in the presence of the ETA receptor antagonist suggesting the role of the ETa receptor in the regulation of the extracellular matrix. ANG II treatment also increased ETB receptor protein levels in a time-dependent manner. ANG II treatment in the presence of the ETB receptor antagonist significantly increased ET-1 peptide levels. On another hand, the ETB receptor agonist, Sarafotoxin 6C, significantly decreased ET-1 peptide levels. These data implicate the role of the ETb receptor in the clearance of the ET-1 peptide. Conclusion: ANG II-induced increases of ET-1 peptide appears to be mediated by reactive oxygen species derived from NAD(P)H oxidase. Both the ETA and ETB receptors are expressed in adventitial fibroblasts. The ETA receptor subtype mediates collagen I expression, while the ETB receptor may play a protective role through increasing the clearance of the ET- 1 peptide.

Primary characterization of genes involved in the colony development of the insect pathogenic fungus Metarhizium anisopliae /

Strain improvement of the insect pathogenic fungus Metarhizium anisopUae is necessary to increase its virulence towards agricultural pests and thus improve its commercial efficacy. Nevertheless, the release of genetically modified conidia in crop fields may negatively affect the ecosystem. Controlling conidiation is a potential means of limiting the release of engineered strains since conidia are the infective propagules and the means of dispersal. The purpose of this study was to research the colony development of M. anisopUae to identify potential targets for genetic manipulation to control conidiation. Following Agrobacterium tumefaciem insertional mutagenesis, phenotypic mutants were characterized using Y-shaped adaptor dependent extension PCR. Four of 1 8 colony development recombinants had T-DNA flanking sequences with high homology to genes encoding known signaling pathway proteins that regulate pathogenesis and/or asexual development in filamentous fungi. Conidial density counts and insect bioassays suggested that a Serine/Threonine protein kinase COTl homolog is not essential for conidiation or virulence. Furthermore, a choline kinase homolog is important for conidiation, but not virulence. Finally, the regulator of G protein signaling CAG8 and a NADPH oxidase NoxA homolog are necessary for conidiation and virulence. These genes are candidates for further investigation into the regulatory pathways controlling conidiation to yield insight into promising gene targets for biocontrol strain improvement.

Mechanisms of endothelin-1 induced reactive oxygen species production in vascular adventitial fibroblasts

With the relationship between endothelin-1 (ET-1) stimulation and reactive oxygen species (ROS) production unknown in adventitial fibroblasts, I examined the ROS response to ET-1 and angiotensin (Ang II). ET-1 -induced ROS peaked following 4 hrs of ET-1 stimulation and was inhibited by an ETA receptor antagonist (BQ 123, 1 uM) an extracellular signal-regulated kinase (ERK) 1/2 inhibitor (PD98059, 10 uM), and by both a specific, apocynin (10 uM), and non-specific, diphenyleneiodonium (10 uM), NAD(P)H oxidase inhibitor. NOX2 knockout fibroblasts did not produce an ET-1 induced change in ROS levels. Ang II treatment increased ROS levels in a biphasic manner, with the second peak occurring 6 hrs following stimulation. The secondary phase of Ang II induced ROS was inhibited by an ATi receptor antagonist, Losartan (100 uM) and BQ 123. In conclusion, ET-1 induces ROS production primarily through an ETA-ERKl/2 NOX2 pathway, additionally, Ang II-induced ROS production also involves an ETa pathway.

The roles of ERK1/2 and PI3K in abnormal vascular functions in angiotensin II-infused hypertensive rats

Background: Ang II plays a major role in cardiovascular regulation. Recently, it has become apparent that vascular superoxide anion may play an important role in hypertension development. Treatment with antisense NAD(P)H oxidase or SOD decreased BP in Ang II-infused rats. Wang et al recently reported mice which lack one of the subunits of NAD(P)H oxidase developed hypertension at a much lower extent when compared to the wild type animals infused with Ang II, indicating that superoxide anion contributes to elevation in BP in the Ang II-infused hypertensive model. In the Ang II-infused hypertensive model, altered reactivity of blood vessels is often associated with the elevation of systolic blood pressure. We have observed abnormal tension development and impaired endothelium-dependent relaxation in the isolated aorta of Ang II-infused and DOCA-salt hypertensive rats. Recently, several other cellular signal molecules, including ERK1I2 and PI3K, have been determined to play important roles in the regulation of smooth muscle contraction and relaxation. ERKl/2 and PI3K pathways are also reported to contribute to Ang II induced cell growth, hypertrophy, remodeling and contraction. Moreover, these signaling pathways have shown ROS-sensitive properties. Therefore, the aim of the present study is to investigate the roles of ERKl12 and PI3K in vascular oxidative stress, spontaneous tone and impaired endothelium relaxation in Ang II-infused hypertensive model. Hypothesis: We hypothesize that the activation of ERKl12 and PI3K are elevated in response to an Ang II infusion for 6 days. The elevated activation of phospho-ERKl/2 and PI3K mediated the increased level of vascular superoxide anion, the abnormal vascular contraction and impaired endothelium-dependent vascular relaxation in Ang II-infused hypertensive rats. Methods: Vascular superoxide anion level is measured by lucigenin chemiluminescence. Spontaneous tone and ACh-induced endothelium-dependent relaxation was measured by isometric tension recording in organ chamber. The activity of ERK pathway will be measured by its Western blot of phosphorylation of ERK. PI3K activity was evaluated indirectly by Western blot of the phosphorylation of PDKl, a downstream protein of PI3K signaling pathway. The role of each pathway was also addressed via comparing the responses to the specific inhibitors. Results: Superoxide anion was markedly increased in the isolated thoracic aorta from Ang II-infused rats. There was spontaneous tone developed in rings from Ang II-induced hypertensive but not sham-operated normotensive rats. ACh-induced endothelium-dependent relaxation function is impaired in Ang II-infused hypertensive rats. Superoxide dismutase and NAD(P)H oxidase inhibitor, apocynin, inhibited the abnormal spontaneous tone and ameliorated impaired endothelium-dependent relaxation. The expression of phopho-ERKII2 was enhanced in Ang II-infused rats, indicating the activity of ERK1I2 could be increased. MEK1I2 inhibitors, PD98059 and U126, but not their inactive analogues, SB203580 and U124, significantly reduced the vascular superoxide anion in aortas from Ang II-infused rats. The MEK1I2 inhibitors reduced the spontaneous tone and improved the impaired endothelium-dependent relaxation in aorta of hypertension. These findings supported the role of ERKII2 signaling pathway in vascular oxidative stress, spontaneous tone and impaired endothelium-dependent relaxation in Ang II-infused hypertensive rats. The amount of phospho-PDK, a downstream protein of PI3K was increased in Ang II rats indicating the activity of PI3K activity was elevated. Strikingly, PI3K significantly inhibited the increase of superoxide anion level, abnormal spontaneous tone and restored endothelium-dependent relaxation in Ang II-infused hypertensive rats. These findings indicated the important role of PI3K in Ang II-infused hypertensive rats. Conclusion: ERKII2 and PI3K signaling pathways are sustained activated in Ang II-infused hypertensive rats. The activated ERKII2 and PI3K mediate the increase of vascular superoxide anion level, vascular abnormal spontaneous tone and impaired endothelium-dependent relaxation.