Peter R. Rich

The sites of superoxide anion generation in higher plant mitochondria.

Abstract A variety of higher plant mitochondria and submitochondrial particles with varying degrees of cyanide insensitivity have been examined for their possible Superoxide anion generating capacity. It was found that neither the cytochrome oxidase nor the alternative oxidase pathways produced significant quantities of Superoxide anions. All preparations examined generated Superoxide anions to a small extent with NADH as respiratory substrate, but almost negligibly with succinate as respiratory substrate. A component of the NADH-supported activity was insensitive to cyanide, antimycin A, and salicylhydroxamic acid. Hence most of this activity is attributed to direct reduction of oxygen by the flavoprotein NADH dehydrogenases. The remainder may be caused by oxygen reduction in the ubiquinone-cytochrome b region of the chain. In some plant mitochondria and submitochondrial particles, a contaminating tyrosinase activity, which can catalyze the oxidation of epinephrine by molecular oxygen, causes a very large interference in Superoxide anion determinations. Methods of distinguishing and measuring these various activities are discussed.

Two Menkes-type ATPases supply copper for photosynthesis in Synechocystis PCC 6803

Synechocystis PCC 6803 contains four genes encoding polypeptides with sequence features of CPx-type ATPases, two of which are now designatedpacS and ctaA. We show that CtaA and PacS (but not the related transporters, ZiaA or CoaT) facilitate switching to the use of copper (in plastocyanin) as an alternative to iron (in cytochrome c 6) for the carriage of electrons within the thylakoid lumen. Disruption of pacSreduced copper tolerance but enhanced silver tolerance, andpacS-mediated restoration of copper tolerance was used to select transformants. Disruption of ctaA caused no change in copper tolerance but reduced the amount of copper cell−1. In cultures supplemented with 0.2 μmcopper, photooxidation of cytochrome c 6 (PetJ) was depressed in wild-type cells but remained elevated in bothSynechocystis PCC 6803(ctaA) andSynechocystis PCC 6803(pacS). Conversely, plastocyanin transcripts (petE) were less abundant in both mutants at this [copper]. Synechocystis PCC 6803(ctaA) and Synechocystis PCC 6803(pacS) showed increased iron dependence with impaired growth in deferoxamine mesylate (iron chelator)-containing media. Double mutants also deficient in cytochromec 6, Synechocystis PCC 6803(petJ,ctaA) and Synechocystis PCC 6803(petJ,pacS), were viable, but the former had increased copper dependence with severely impaired growth in the presence of bathocuproinedisulfonic acid (copper chelator). Analogous transporters are likely to supply copper to plastocyanin in chloroplasts.

The kinetics and thermodynamics of the reduction of cytochrome c by substituted p-benzoquinols in solution

1. The mechanisms by which p-benzoquinol and its derivatives reduce cytochrome c in solution have been investigated. 2. The two major reductants are the species QH- (anionic quinol) and Q.- (anionic semiquinone). A minor route of electron transfer from the fully protonated QH2 species can also occur. 3. The relative contributions of these routes to the overall reduction rate are governed by pH, ionic strength and relative reactant concentrations. 4. For a series of substituted p-benzoquinols, the forward rate constant, k1, of the anionic quinol-mediatd reaction is related to the midpoint potential of the QH-/QH. couple involved in the rate-limiting step, as predicted by the theory of Marcus for outer-sphere electron transfer reactions in a bimolecular collision process. 5. A mechanism for the biological quinol oxidation reactions in mitochondria and chloroplasts is proposed based upon the findings with these reactions in solution.

Proton uptake by cytochrome c oxidase on reduction and on ligand binding

On reduction, cytochrome oxidase was found to take up 2.4 +/- 0.1 protons in the pH range 7.2-8.5, of which 2 are associated with the binuclear centre, and the remaining fractional proton with haem a/CuA. Ligation to oxidised cytochrome oxidase of the azide, formate, fluoride or cyanide anions is accompanied by uptake of one proton. In the case of the reduced enzyme, no protonation changes are observed on binding O2 (Hallén S. and Nilsson T. (1992) Biochemistry 31, 11853-11859) or CO. Cyanide binding to reduced oxidase is, in contrast, still accompanied by uptake of a proton. These findings are discussed in terms of our previously-published proposal for the ligand chemistry of the binuclear site. The results overall suggest a principle of electroneutrality of redox and ligand state changes of the binuclear centre, with charge compensations provided only by protonation reactions.

Studies on the mechanism of inhibition of redox enzymes by substituted hydroxamic acids.

Substituted primary hydroxamic acids were found to inhibit the catalytic activity of a number of redox enzymes. The inhibition was not related to the nature of the metal-active site of the enzyme nor to the nature of the oxygen-containing substrate. Two easily available enzymes, mushroom tyrosinase (monophenol,dihydroyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which were potently inhibited by hydroxamic acids, were chosen for more detailed study. A kinetic analysis of the inhibitory effects on the partially purified tyrosinase of mushroom (Agaricus bispora) revealed that inhibition was reversible and competiitive with respect to reducing substrate concentration, but was not competitive with respect to molecular oxygen concentration. A spectrophotometric and EPR study of the binding of salicylhydroxamic acid to horseradish peroxidase revealed that his hydroxamic acid was bound to the enzyme in the same manner as a typical substrate, hydroquinone. Spectroscopic and thermodynamic measurements of the binding reactions suggested that this binding site is close, to but, not directly onto, the heme group of the enzyme. From these results it is concluded that the mode of inhibition of hydroxamic acid need not be, as generally supposed, by metal chelation, and mechanisms involving either hydrogen bonding at the reducing substrate binding site or the formation of a charge transfer complex between hydroxamic acid and an electron-accepting group in the enzyme are considered to be more feasible. The relevance of these findings to deductions on the nature of other hydroxamic acid-inhibitable systems is discussed.

The redox potentials of the b-type cytochromes of higher plant chloroplasts.

1. In fresh chloroplasts,three b-type cytochromes exist. These are b-559HP (lambda max, 559 nm; Em at pH 7, +370 mV; pH-independent Em), b-559LP (lambda max, 559 nm; Em at pH 7, +20 mV; pH-independent Em) and b-563 (lambda max, 563 nm; Em at pH 7, -110 mV; pH-independent Em), b-559HP may be converted to a lower potential form (lambda max, 559 nm; Em at pH 7, +110 mV; pH-independent Em). 2. In catalytically active b-f particle preparations, three cytochromes exist. These are cytochrome f (lambda max, 554 nm; Em at pH 7, +375 mV, pK on oxidised cytochrome at pH 9), b-563 (lambda max, 563 nm; Em at pH 7, -90 mV, small pH-dependence of Em) and a b-559 species (lambda max, 559 nm, Em at pH 7, +85 mV; pH-independent Em). 3. A positive method of demonstration and estimation of b-559LP in fresh chloroplasts is described which involves the use of menadiol as a selective reductant of b-559LP.

A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition

A bacterial two-hybrid assay revealed interaction between a protein now designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA (cellular import) and PacS (thylakoid import) but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803). The specificity of metallochaperone interactions coincides with metal specificity. After reconstitution in a N2 atmosphere, bacterial Atx1 bound 1 mol of copper mol−1, and apoPacSN acquired copper from copper-Atx1. Copper was displaced from Atx1 byp-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacSN. This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. In copper super-supplemented mutants, photooxidation of cytochrome c 6 was greater in Δatx1ΔctaA than in ΔctaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c 6 to copper in plastocyanin for photosynthetic electron transport. Cytochrome oxidase activity was also less in membranes purified from low [copper]-grown Δatx1 or ΔpacS, compared with wild-type, but the double mutant Δatx1ΔpacS was non-additive, consistent with Atx1 acting via PacS. Conversely, activity in Δatx1ΔctaA was less than in either respective single mutant, revealing that Atx1 can function without the major copper importer and consistent with a role in recycling endogenous copper.

Quinol oxidation in Arum maculatum mitochondria and its application to the assay, solubilisation and partial purification of the alternative oxidase

The nature of the alternative oxidase which is present in a variety of higher plant mitochondria [l] has remained elusive. This stems from the fact that the oxidase is indistinct both in its spectrophotometric [2] and in its electron paramagnetic resonance parameters [3,4]. It can be shown, however, that the oxidase itself can accept electrons from the ubiquinone of the normal electron-transport pathway, which therefore forms the branchpoint for electron flow [5.6] . A further problem encountered in attempts at characterisation of the terminal components is the apparent lability of the oxidase [7] and the lack of an artificial donor which may be used for the direct assay of the alternative oxidase. Investigations which utilise menadiol and ubiquinol-I as possible donors to the alternative oxidase of Arum maculatum mitochondria are presented here. It is demonstrated that these quinols donate electrons to a point which is at or very close to the alternative oxygen-consuming step. Further, the oxidase may be detergent-solubilised and purified to some extent when these quinols are employed as donors, without rapid loss of activity. A preliminary communication of this work has already been presented (81 .

ON THE MECHANISM OF QUINOL OXIDATION IN THE BC1 COMPLEX

The question of whether significant levels of a semiquinone can be generated in the Qo site of thebc 1 complex under conditions of oxidant-induced reduction is relevant to the mechanism of bifurcation of electron transfer in this site. It has already been reported that beef heart submitochondrial particles under such conditions exhibit an EPR-detectable semiquinone, which is distinct from Q⨪i and which was attributed to a semiquinone in the Qo site (de Vries, S., Albracht, S. P. J., Berden, J. A., and Slater, E. C. (1981) J. Biol. Chem. 256, 11996–11998). However, we show here that this signal, which can be generated to a level of around 0.1 perbc 1 monomer, is insensitive to the Qo site inhibitors myxothiazol, E-β-methoxyacrylate-stilbene, and stigmatellin, indicating that it does not arise from a Q⨪o species. Based on sensitivities to inhibitors of other Q sites, up to 60% of the signal may arise from semiquinones of complexes I and II. We further show that the iron-sulfur center remains EPR silent under oxidant-induced reduction conditions. Overall, the results indicate that, under conditions of oxidant-induced reduction, the Qo site is occupied primarily by quinol with the iron-sulfur center oxidized, or, possibly, by an antiferromagnetically coupled semiquinone/reduced iron-sulfur center pair, which are EPR silent. This is discussed in relation to proposed mechanisms of quinol oxidation in the Qo site, and we describe a minimal intermediate-controlled bifurcation model based on rate constants by which bifurcated electron transfer at the Qo site might occur.

The mitochondrial respiratory chain

In the present chapter, the structures and mechanisms of the major components of mammalian mitochondrial respiratory chains are reviewed. Particular emphasis is placed on the four protein complexes and their cofactors that catalyse the electron transfer pathway between oxidation of NADH and succinate and the reduction of oxygen to water. Current ideas are reviewed of how these electron transfer reactions are coupled to formation of the proton and charge gradient across the inner mitochondrial membrane that is used to drive ATP synthesis. Additional respiratory components that are found in mammalian and plant, fungal and algal mitochondria are also reviewed.