J.B. Jackson

Proton-translocating transhydrogenase and NAD- and NADP-linked isocitrate dehydrogenases operate in a substrate cycle which contributes to fine regulation of the tricarboxylic acid cycle activity in mitochondria

H+‐transhydrogenase (H+‐Thase) and NADP‐linked isocitrate dehydrogenase (NADP—ICDH) are very active in animal mitochondria but their physiological function is only poorly understood. This is especially so in the case of the heart and muscle, where there are no major consumers of NADPH. We propose here that H+‐Thase and NADP—ICDH have a combined function in the fine regulation of the activity of the tricarboxylic acid (TCA) cycle, providing enhanced sensitivy to changes in energy demand. This is achieved through cycling of substrates by NAD‐linked ICDH, NADP‐linked ICDH and H+‐Thase. It is proposed that NAD—ICDH operates in the forward direction of the TCA cycle, but NADP—ICDH is driven in reverse by elevated levels of NADPH resulting from the action of the transmembrane proton electrochemical potential gradient (Δp) on H+‐Thase. This has the effect of increasing the sensitivity to allosteric modifiers of NAD—ICDH (NADH, ADP, ATP, Ca2+ etc), potentially giving rise to large changes in the net flux from iso‐citrate to α‐ketoglutarate. Furthermore, changes in the level of Δp resulting from changes in the demand for ATP would, via H+‐Thase, shift the redox state of the NADP pool and this, in turn, would lead to a change in the rate of the reaction catalysed by NADP—ICDH and hence to an additional and complementary effect on the net metabolic flux from isocitrate to α‐ketoglutarate. Other consequences of this substrate cycle are, (i) the production of heat at the expense of Δp, which may contribute to thermoregulation in the animal, and (ii) an increased rate of dissipation of Δp (leak).

The relation between membrane ionic current and atp synthesis in chromatophores from rhodopseudomonas capsulata

Abstract (1) Under conditions in which membrane potential (Δψ) was the sole contributor to the proton-motive force, the steady-state rate of ATP synthesis in chromatophores increased disproportionately when Δψ was increased: the rate had an approximately sixth-power dependence on Δψ. (2) Simultaneous measurements showed that the dissipative ionic current (JDIS) across the chromatophore membrane had a related dependence on Δψ, i.e., the membrane conductance increased markedly as Δψ increased. (3) For comparable Δψ values, JDIS was greater in phosphorylating than in non-phosphorylating chromatophores. For comparable actinic light intensities, Δψ was smaller in phosphorylating than in non-phosphorylating chromatophores. (4) At either low pH or in the presence of venturicidin, oligomycin or dicyclohexylcarbodiimide to inhibit ATP synthesis, JDIS was substantially depressed, particularly at high Δψ. Even under these conditions the membrane conductance was dependent on Δψ. (5) Also in intact cells, JDIS was depressed in the presence of venturicidin. Points 1–5 are interpreted in terms of a Δψ -driven H+ flux through the F0 channel of the ATPase synthase. The high-power dependence of the F0 conductance on Δψ determines the dependence of the rate of ATP synthesis on Δψ. The Δψ -dependent conductance of F0 dominates the electrical properties of the membrane. In chromatophores the ionic current accompanying ATP synthesis was more than 50% of the total membrane ionic current at maximal Δψ. (6) The rate of cyclic electron transport was calculated from JDIS. This led to an estimate of 0.77 ± 0.22 for the ATP 2 e − ratio and of 3.5 ± 1.3 for the H + ATP ratio. (7) Severe inhibition of the electron-transport rate by decreasing the light intensity led to an almost proportionate decrease in the rate of ATP synthesis. The chromatophores were able to maintain proportionality by confining electron-transport phosphorylation to a narrow range of Δψ. This is a consequence of the remarkable conductance properties of the membrane.

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification, characterisation and distinction from a single reductase for trimethylamine-N-oxide, dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR+ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.

Periplasmic location of the terminal reductase in trimethylamine N-oxide and dimethylsulphoxide respiration in the photosynthetic bacterium Rhodopseudomonas capsulata

Abstract (1) The trimethylamine N -oxide and dimethylsulphoxide reductase activities of Rhodopseudomonas capsulata have been shown to have a periplasmic location. Previous evidence for a cytoplasmic location was based on an observation of a soluble FMN-dependent NADH-trimethylamine N -oxide oxidoreductase activity in soluble total cell extracts (Cox, J.C., Madigan, M., Favinger, J.F., Gest, H. (1980) Arch. Biochem. Biophys. 204, 10–17). The latter activity has now been shown to arise from the combination of a cytoplasmic NADH-FMN oxidoreductase and the periplasmic dihydro: FMN-trimethylamine N -oxide oxidoreductase in such total extracts. A periplasmic location is consistent with the linking of trimethylamine N -oxide reductase to the proton-translocating electron-transport chain as concluded earlier (McEwan, A.G., Ferguson, S.J. and Jackson, J.B. (1983) Arch. Microbiol. 136, 300–305). (2) Observations on the relative dimethylsulphoxide and trimethylamine N -oxide reductase activities in extracts from cells grown on either of these electron acceptors, together with the identification of an identical electrophoretic mobillity of both activities on a polyacrylamide gel, suggest that a single enzyme is probably responsible for both activities in Rps. capsulata .

Interconversion of two kinetically distinct states of the membrane-bound and solubilised H+-translocating ATPase from Rhodospirillum rubrum

The ATP synthetase enzymes which catalyse the terminal reactions of oxidative and photophosphorylation may be dislodged from their membrane vesicles by a variety of techniques. Studies on ATPase activity of the solubilised enzymes will only provide information on the phosphorylation mechanism if a clear relationship with the membrane-bound enzymes can be established. In fact many differences in behaviour between membrane-bound and solubilised enzyme have been observed (see review by Pederson [l] ). The ATPase activity of Rhs. rubrum chromatophores is dependent on the presence of either Ca2+ or Mg2+ [2,3] . In contrast, the solubilised coupling factor (Fi-type) is strictly Ca”-dependent and is competitively inhibited by Mg2+ [4] . A similar change in divalent cation requirement takes place upon resolution of the chloroplast ATPase [ 5-81 and has been described as allotopic [7], implying that the substrate specificity of the enzyme is modified by membrane binding [9 ] . The experiments described in this paper show that the divalent cation requirement of the solubilised enzyme may be duplicated in the membrane-bound system provided that the chromatophores are treated with high concentrations of uncoupling agent. The conclusion is that the substrate specificity is determined, not by association with membrane components but by the level of the high-energy state across

Evidence that the ionic conductivity of the cytoplasmic membrane of Rhodopseudomonas capsulata is dependent upon membrane potential

In mitochondria, chloroplasts and bacteria, a protonmotive force [ 1] (A&+) across the coupling membrane is believed to be an energetic intermediate in electron-transport phosphorylation. The dependence of the magnitude of Aj&I+ upon the rate of electron transport is usually non-linear [2-6]. Here, I show that in intact cells of photosynthetic bacteria a voltage-dependent ionic conductance [2] probably accounts for this. membrane conductivity. In [5] relation between the rate of photosynthetic electron transport and ApH in thylakoid membranes was found to ,be non-linear. It was concluded that the ionic conductance of the membrane was dependent on ApH [S]. In [4] data was obtained with mitochondria that were not compatible with this general explanation. Slip in the electron-transport proton pumps (variable n in eq. (1)) was proposed to provide a better explanation [4].

The role of auxiliary oxidants in the maintenance of a balanced redox poise for photosynthesis in bacteria

Abstract Carotenoid absorbance changes were used to monitor the development of membrane potential in intact cell suspensions of Rhodopseudomonas capsulata strain N22. Low concentrations of phenazine methosulphate almost completely inhibited the generation of membrane potential in the light by anaerobic cells. The light-dependent reactions were restored by addition of either trimethylamine N -oxide, dimethylsulphoxide, nitrous oxide, or oxygen. In Rhodopseudomonas capsulata strain N22 DNAR + addition of nitrate was also effective. The inhibition by phenazine methosulphate and restoration by auxiliary oxidant were observed in the presence of sufficient rotenone to block NADH dehydrogenase or with low concentrations of uncoupling agent to dissipate the membrane potential under dark, anaerobic conditions. It is suggested that in intact cells of these organisms there are mechanisms which operate to maintain the electron-transport chain at an optimal redox poise for efficient photosynthesis. Phenazine methosulphate perturbs the optimal redox poise by hastening equilibrium of the photosynthetic electron-transport chain with low-potential couples in the cell. The addition of auxiliary oxidants restores the optimal redox poise. This suggests a role in photosynthesis for the pathways of respiratory electron flow to nitrate, nitrous oxide, trimethylamine N -oxide/dimethylsulphoxide and oxygen.

Evidence That the Transfer of Hydride Ion Equivalents between Nucleotides by Proton-translocating Transhydrogenase Is Direct

The molecular masses of the purified, recombinant nucleotide-binding domains (domains I and III) of transhydrogenase from Rhodospirillum rubrum were determined by electrospray mass spectrometry. The values obtained, 40,273 and 21,469 Da, for domains I and III, respectively, are similar to those estimated from the amino acid sequences of the proteins. Evidently, there are no prosthetic groups or metal centers that can serve as reducible intermediates in hydride transfer between nucleotides bound to these proteins. The transient-state kinetics of hydride transfer catalyzed by mixtures of recombinant domains I and III were studied by stopped-flow spectrophotometry. The data indicate that oxidation of NADPH, bound to domain III, and reduction of acetylpyridine adenine dinucleotide (an NAD+ analogue), bound to domain I, are simultaneous and very fast. The transient-state reaction proceeds as a biphasic burst of hydride transfer before establishment of a steady state, which is limited by slow release of NADP+. Hydride transfer between the nucleotides is evidently direct. This conclusion indicates that the nicotinamide rings of the nucleotides are in close apposition during the hydride transfer reaction, and it imposes firm constraints on the mechanism by which transhydrogenation is linked to proton translocation.

The role of c-type cytochromes in the photosynthetic electron transport pathway of Rhodobacter capsulatus

(1) Short flash excitation of membrane vesicles of a cytochrome-c2-deficient mutant of Rhodobacter capsulatus (strain MT-G4/S4) led to rapid oxidation of a c-type cytochrome. In redox titrations, the photooxidation of c-type cytochrome was attenuated with a midpoint of approx. +360 mV. Vesicles from a control strain, MT1131, gave similar results. These findings are consistent with those of Prince et al. (Prince, R.C., Davidson, E., Haith, L.E. and Daldal, F. (1986) Biochemistry 25, 5208-5214). (2) In anaerobic intact cells the extent of rapid re-reduction of c-type cytochrome oxidised after a flash was less in MT-G/S4 than in MT1131. Cytochrome c reduction in both strains was inhibited by myxothiazol. The myxothiazol-sensitive component of the electrochromic absorbance change in cells indicated that rapid charge separation through the cytochrome bc1 complex was less extensive after a flash in MT-G4/S4 than in MT 1131. (3) In anaerobic intact cells and in chromatophores of Rb. capsulatus strain MT-GS18, a mutant deficient in both cytochrome c1 and cytochrome c2, flash excitation led to the oxidation of c-type cytochrome. Redox titrations and spectra of chromatophores suggested that this is the same cytochrome as was photooxidized in vesicles of MT-G4/S4 and MT1131. This result is in contrast with earlier findings (Prince, R.C. and Daldal, F. (1987) Biochim. Biophys, Acta 894, 370-378) in which it was reported that no photooxidation of c-type cytochrome occurred in the absence of c1 and c2, and argues against the possibility that cytochrome c1 can rapidly and directly donate electrons to the reaction centre. (4) It is proposed that a previously uncharacterized, membrane-bound c-type cytochrome (Em7 approximately +360 mV) is present in Rb-capsulatus MT1131, in the c2-deficient mutant MT-G4/34 and in the c1/c2-deficient mutant MTGS18. This cytochrome and cytochrome c2 are alternative electron donors to the reaction centre in strain MT1131.

In vivo redox poising of the cyclic electron transport system of Rhodobacter capsulatus and the effects of the auxiliary oxidants, nitrate, nitrous oxide and trimethylamine N-oxide, as revealed by multiple short flash excitation

Abstract Intact cells of Rhodobacter capsulatus in the presence of myxothiazol were exposed to trains of short flashes of saturating light and the pattern of the absorbance changes due to P870, cytochrome ( c 1 + c 2 ) and the carotenoids that report on the membrane potential were monitored. Myxothiazol inhibits cyclic electron transport and therefore the extent to which electron donors and acceptors of the reaction centre are available for photochemistry is revealed. In darkened anaerobic suspensions of cells in the presence of myxothiazol, only the first two flashes in the train led to charge separation in the photosynthetic reaction centres. The results indicated that the quinone pool and quinone bound at the Q B site in the reaction centre were extensively reduced and quinone bound at Q A was partly reduced before initiation of flash excitation. Thus under these conditions, and in the absence of myxothiazol, cyclic electron transport would be restricted. In the presence of oxygen or the auxiliary oxidants trimethylamine N -oxide, NO − 3 or N 2 O, the oxidation/reduction reactions and the electrochromic absorbance changes suggested that the pool and reaction centre quinones became more oxidised. Thus, the system was poised at a potential more conducive to optimal rates of photosynthetic electron transport. By reference to experiments on the growth of Rb. capsulatus (Richardson, D.J., King, G.F., Kelly, D.J., McEwan, A.G., Ferguson, S.J. and Jackson, J.B. (1988) Arch. Microbiol. 150, 131–137), it is argued that redox poising by the auxiliary oxidants is physiologically important, especially at low light intensities. Flash train experiments reveal that over-reduction of the quinones is more severe with succinate as a carbon source than with malate and this accounts for the observation that the rate of growth on succinate is decreased more strongly at low light intensities.