On the conformation of reconstituted ferredoxin:NADP oxidoreductase in the thylakoid membrane. Studies via triplet lifetime and rotational diffusion with eosin isothiocyanate as label

Eosin isothiocyanate was covalently bound to isolated ferredoxin-NADP⁺ reductase under protection of the NADP-binding domain. The bound label did not impair the functional reconstitution of the enzyme into depleted thylakoid membranes. Laser spectrophotometric experiments were carried out on thylakoids which were reconstituted with labeled ferredoxin-NADP⁺ reductase. Bound eosin isothiocyanate was used as a spectroscopic probe for conformational changes of ferredoxin-NADP⁺ reductase in either of two ways: We studied the rotational diffusion of labeled ferredoxin-NADP⁺ reductase in the membrane by the photoselection technique, and we studied the triplet lifetime of bound eosin, which measures polypeptide chain flexibility (via access of oxygen) around the binding site. The latter technique was complemented by measurements of the librational motion of bound dye. We observed: (1) When ferredoxin is absent, ferredoxin-NADP⁺ reductase undergoes very rapid rotational diffusion in the thylakoid membrane (correlation time less than 1 μs at 10°C). This is drastically slowed down (40 μs) upon addition of water-soluble ferredoxin. We propose that ferredoxin mediates the formation of a ternary complex with ferredoxin-NADP⁺ reductase and the Photosystem I complex. According to our data, this complex would live longer than required for the photoreduction of ferredoxin-NADP⁺ reductase by Photosystem I via ferredoxin. (2) Under the given incubation conditions, the binding sites for eosin isothiocyanate were located in the FAD domain of ferredoxin-NADP⁺ reductase. We found increased chain flexibility in this domain upon addition of NADP. This suggests induced fit for the binding of NADP and allosteric control of the FAD domain by the remote NADP domain. (3) Acidification of the internal phase of thylakoids decreased the chain flexibility in the FAD domain. This is of particular interest, since ferredoxin-NADP⁺ reductase is a peripheral external membrane protein. It suggests the existence of a binding protein for the oxidoreductase which spans the membrane and senses the internal pH     

Chemical modification of the active site of ferredoxin-NADP reductase and conformation of the binary ferredoxin/ferredoxin-NADP reductase complex in solution

Ferredoxin-NADP⁺ reductase (EC 1.18.1.2) was chemically modified by the triplet probe eosin isothiocyanate (eosin-NES). Incorporation of 1 mol eosin-NCS/mol ferredoxin-NADP⁺ reductase completely inhibited binding of NADP⁺/NADPH to the enzyme. Binding of eosin without the reactive group to the enzyme was shown to be reversible but to compete with NADP⁺/NADPH with a K_i of approx. 5 μM. The binding site of eosin-NCS has been located in the primary sequence ferredoxin-NADP⁺ reductase. After specific cleavage of arginine with trypsin a single labelled peptide was obtained and identified as the fragment from residue 179–228 in the primary sequence. Binding of eosin-NCS occurred in either of two predicted helices (residues 179–189 or 212–228) which are both part of an /β structure characteristic for nucleotide binding folds. The rotational diffusion in solution of the eosin-labelled ferredoxin-NADP⁺ reductase and its complex with ferredoxin was measured with laser flash spectroscopy under photoselection. From the measured rotational correlation times and the known structure of ferredoxin-NADP⁺ reductase at 3.7 Å resolution, we propose that ferredoxin is bound to ferredoxin-NADP⁺ reductase between the two domains of the flavoprotein. The two ferredoxin-NADP⁺ reductase domains and ferredoxin form a triangle which results in a highly integrated binary complex.     

Fluorescence induction and activity of ferredoxin-NADP reductase in Bryopsis chloroplasts

Effects of medium osmolarity on the rate of CO₂ fixation, the rate of the NADP⁺-Hill reaction, and the DPS₁ transient of chlorophyll fluorescence were measured in intact Bryopsis chloroplasts. Upon decreasing the sorbitol concentration from 1.0 M (the isoosmotic conditions) to 0.25 M, the envelopes of the chloroplasts became leaky to small molecules, resulting in a considerable depression of the CO₂-fixation rate and a higher rate of the NADP⁺-Hill reaction whereas the DPS₁ transient was unaffected. This DPS₁ transient of chlorophyll fluorescence is thought to be caused by the photoactivation of electron flow on the reducing side of Photosystem I at a site occurring after ferredoxin and probably before the reduction of NADP⁺ (Satoh, K. and Katoh, S. (1980) Plant and Cell Physiol. 21, 907–916). Little effect of NADP⁺ on the DPS₁ transient and a marked lag in NADP⁺ photo-reduction in dark-adapted (inactivated) chloroplasts support the hypothesis that the site of dark inactivation is prior to the reduction site of NADP⁺, and therefore, that ferredoxin-NADP⁺ reductase is inactivated in the dark and activated in the light. Moreover, at 0.25 M sorbitol, the activity of ferredoxin-NADP⁺ reductase itself (2,6-dichlorophenolindophenol reduction by NADPH) was shown to increase according to dark-light transition of the chloroplasts. At low osmolarities (below 0.1 M sorbitol), the difference in the diaphorase activity between dark-and light-adapted chloroplasts and the lag time observed in the NADP⁺ photoreduction were lowered. This may correspond to a less pronounced DPS₁ transient at low concentrations of sorbitol. The mechanism of the photo-activation is discussed.     

Isolation and properties of chloroplast particles of Scenedesmus obliquus D3 with high photochemical activity

An improved and standardized procedure for isolation of chloroplast particles from the unicellular green alga, Scenedesmus obliquus, D₃, is described. The method is generally applicable to heterotrophically- and autotrophically-grown cells of Scenedesmus as well as to Chlamydomonas reinhardti and Chlorella sorokiniana (7-11-05) cultures. Chloroplast particles with high NADP⁺ photoreducing capacity are obtained from heterotrophic cultures only when the cell types are random and the culture is in the logarithmic growth phase; maximal rates of 240–260 μmoles NADP⁺ reduced/h per mg chlorophyll are achieved. Optimal conditions for separation of such chloroplast particles require the use of Tricine buffer (20 mM, pH 7.5), 50 nM EDTA, 10 mM KCl and 0.5 mM dithiothreitol in the breaking medium; for the maintenance of high photochemical activity it is necessary to store particles in a solution consisting of 0.4 M sucrose, 30 mM KCl and 1% bovine serum albumin.

Optimum reaction conditions were developed and the properties of the isolated particles investigated. Maximal activities are obtained when the sucrose concentration is maintained below 0.4 M; the pH optimum with Tricine buffer is between 7.8–8.1; and at least 30 mM Cl⁻ is required. Red actinic light (wavelength >620 nm) with an intensity of 10⁶ ergs/cm² per s is required for saturation.

Ferrodoxin and ferredoxin-NADP⁺ oxidoreductase are lost from the particles during the preparatory procedures and maximum photochemical activity is attained only when they are added back in balanced amounts. Stimulatory effects of added plastocyanin and cytochrome c-553 are noted only with particles having an initially low photochemical activity.     

The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity

Evidence has been obtained that NADPH may serve as a physiological source of reducing power for nitrogenase activity in Azotobacter vinelandii. NADH was ineffective. Electron transfer from NADPH to nitrogenase depended on four factors native to A. vinelandii cells: azotobacter ferredoxin, azotoflavin, a component replaceable by spinach ferredoxin-NADP⁺ reductase and another soluble, heat-labile component not yet chemically characterized. The four factors probably constitute an electron transport chain between NADPH and nitrogenase.     

Photogeneration of singlet oxygen (1O2) and free radicals (Sen·-, O·-2) by tetra-brominated hypocrellin B derivative

To improve photodynamic activity of the parent hypocrellin B (HB), a tetra-brominated HB derivative (compound 1) was synthesized in high yield. Compared with HB, compound 1 has enhanced red absorption and high molar extinction coefficients. The photodynamic action of compound 1, especially the generation mechanism and efficiencies of active species (Sen^·-, O^·-₂ and ¹O₂) were studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of compound 1, the semiquinone anion radical of compound 1 is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of compound 1. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from Sens^·- to the ground state molecular oxygen. The efficiencies of Sens^·- and O^·-₂ generation by compound 1 are about three and two times as much as that of HB, respectively. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet compound 1 to ground state oxygen molecules. The quantum yield of singlet oxygen (¹O₂) is 0.54 in CHCl₃ similar to that of HB. Furthermore, it was found that the accumulation of Sens^·- would replace that of O^·-₂ or ¹O₂ with the depletion of oxygen in the sealed system.     

Influence de l'oxygene sur les transferts d'electrons de la photosynthese. I. Influence de diverses concentrations d'oxygene sur quel ques reactions de Hill

Influence of oxygen on the electron transfers of photosynthesis. I. Influence of some oxygen concentrations on some Hill reactions

The influence of O₂ concentrations on the Hill reactions in the presence of p-benzoquinone, ferricyanide, NADP⁺, NADP⁺ plus ferredoxin has been studied with isolated spinach chloroplasts.

Because of the partial reoxidation of the hydroquinone, which is depending upon the O₂ concentration, it does not seem possible to localize a site of action for O₂.

With ferricyanide the influence of O₂ is weak. However, the rate of ferricyanide reduction is increased in the presence of O₂. The observed stimulation is greater for 21% O₂ than for 70% O₂. Bicarbonate stimulates the ferricyanide reduction and decreases the stimulating effect of 21% O₂.

O₂ decreases the rate of NADP⁺ reduction. Ferredoxin as well as bicarbonate stimulate the NADP⁺ reduction and reduce the O₂ inhibition.

These results seem to indicate that O₂ may enter the electron transport chain at a site situated near Photosystem I and before the ferredoxin's site.

The inhibitory effect of O₂ on the Hill reactions with p-benzoquinone and NADP⁺ is depending upon the plants' growth conditions. It is greater with plants grown under weak light.     

Photosensitization mechanism of active species by the complex of hypocrellin B with aluminum ion

To improve the water solubility and red absorption of the parent hypocrellin B (HB), the complex of HB with aluminum ion has been first synthesized in high yield. The photodynamic action of Al³⁺-HB, especially the generation mechanism of active species, ([Al³⁺-HB]^·-, O^·-₂ and ¹O₂) was studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of Al³⁺-HB, the semiquinone anion radical of Al³⁺-HB is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of Al³⁺-HB. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from [Al³⁺-HB]^·- to the ground state molecular oxygen. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet Al³⁺-HB to ground state oxygen molecules. Furthermore, it is very significant that the accumulation of [Al³⁺-HB]^·- would replace that of O^·-₂ or ¹O₂ with the consumption of oxygen in the sealed system.     

Mitochondrial functions under hypoxic conditions: The steady states of cytochrome c reduction and of energy metabolism

1. The effect of low oxygen concentration on the oxidation-reduction states of cytochrome c and of pyridine nucleotide, on Ca²⁺ uptake, on the energy-linked reduction of pyridine nucleotide by succinate, and on the rate of oxygen consumption have been examined under various metabolic conditions, using pigeon heart mitochondria.

2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50_c) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50_c value with increasing respiratory rate.

3. The fraction of the normoxic respiration that is observed at p50_c is 70–90% under State 4 conditions, but is 30% under State 3 conditions.

4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50_PN) varies less than p50_c, being 0.08 μM in State 3 and 0.06 μM in the uncoupled state.

5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca²⁺ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50_PN).     

The mechanism of photosynthetic sulfate reduction by isolated chloroplasts

The reduction of sulfate by isolated spinach chloroplasts was studied. A reconstituted system of broken chloroplasts and of chloroplast extract reduced sulfate to sulfite in the light when ADP, NADP⁺, ferredoxin and glutathione were added. The chloroplast extract reduced sulfate to sulfite in the dark if supplemented with ATP and with reduced glutathione. Neither ferredoxin nor NADPH were needed for this reduction in the dark.

A sulfite reductase was purified from spinach leaves. Broken chloroplasts and sulfite reductase reduced sulfite to sulfide in the light when ferredoxin was added. NADP⁺ was not required for this reduction.

The results suggest that in chloroplasts a sulfate activated by ATP (phosphoadenosine phosphosulfate) is reduced to sulfite by a sulfhydryl compound and that sulfite is reduced to sulfide by a ferredoxin-dependent sulfite reductase.     

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.     

Calcium and photosynthetic oxygen evolution in cyanobacteria

Calcium activation of oxygen evolution from French-press preparations of Phormidium luridum is largely reversible upon removal of added Ca²⁺. Activation occurs via a first-order binding with a dissociation constant of 2.8 mM. An 8-fold increase in oxygen evolution rate observed upon Ca²⁺ addition is accounted for by a 4-fold increase in the number of active photosynthetic units, and a doubling of turnover rate. While both Ca²⁺ and Mg²⁺ stimulate turnover, unit activation is Ca²⁺ specific. Under optimal conditions, 30% of the units functioning in the intact cell can be recovered in the Ca²⁺-activated preparation.

The Ca²⁺ requirement of P. luridum preparations is not relieved by proton-carrying uncouplers, or by rate-saturating concentrations of the Hill acceptor, ferricyanide. Taken together with the reported stimulation by Ca²⁺ of oxygen evolution in the presence of DCMU (Piccioni, R.G. and Mauzerall, D.C. (1976) Biochim. Biophys. Acta 423, 605–609) these observations strongly suggest a site of Ca²⁺ action within Photosystem II.

The pronounced specificity of the Ca²⁺ requirement appears in preparations of other cyanobacteria (Anabaena flos-aquae and Anacystis nidulans) but not in the eucaryote Chlorella vulgaris. While milder cell-disruption methods bring about some Ca²⁺ dependence in P. luridum, French-press treatment is required for maximal expression of Ca²⁺-specific effects. French-press breakage causes a release of endogenous Ca²⁺ from cells, supporting the view that added Ca²⁺ restores oxygen evolution by satisfying a physiological requirement for the cation.     

The role of polyglucose in oxygen-dependent respiration by a new strain of Desulfovibrio salexigens

Abstract: Desulfovibrio salexigens strain Mastl was isolated from the oxic/anoxic interface of a marine sediment. Growth under sulfate-reducing conditions was accompanied by polyglucose accumulation in the cell with every substrate tested. Highest polyglucose storage was found with glucose (0.8–1.0 g polyglucose (g protein)⁻¹), but the growth rate with this substrate was very low (0.015 h⁻¹). Anaerobically grown cells of strain Mastl exhibited immediate oxygen-dependent respiration. The endogenous oxygen reduction rate was proportional to the polyglucose content. The rate of aerobic respiration of pyruvate was also directly related to the polyglucose content indicating that this organism was only able to respire with oxygen as long as polyglucose was present. Maximum oxygen reduction rates were found at air saturating concentrations and were relatively low (3–50 nmol O₂ min⁻¹ (mg protein)⁻¹). Catalase was constitutively present in anaerobically grown cells. When batch cultures were exposed to oxygen, growth ceased immediately and polyglucose was oxidized to acetate within 40–50 h. Like the oxygen reduction activity, the nitro blue tetrazolium (NBT)-reduction activity in these cells was proportional to the polyglucose content. Under anaerobic starvation conditions there was no correlation between the NBT-reduction activity and polyglucose concentration and polyglucose was degraded slowly within 240 h. The ecological significance of aerobic polyglucose consumption is discussed.     

A high-potential redox component located within cyanobacterial Photosystem II

The calcium-dependent oxygen evolution activity of preparations of Phormidium luridum shows a marked selectivity in favor of ferricyanide over benzoquinone as Hill oxidant. In addition, the rate of oxygen evolution increases with increasing solution redox potential over the range +350 to +550 mV vs. the standard hydrogen electrode. These properties pertain to both 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive and -insensitive fractions of the total oxygen evolution activity. Neither changes in solution potential nor use of oxidants other than ferricyanide obviate the need for added Ca²⁺.

To explain these observations, two models are proposed, each of which invokes the existence of a redox component located within Photosystem II and having a midpoint potential greater than +450 mV. In one model, the postulated species is a donor which competes with water for oxidizing equivalents generated by System II. In the other model, the 450 mV species is a high-potential primary acceptor of System II electrons.     

Electron Transfer Reactions in RB90745, A Bioreductive Drug Having Both Aromatic N-Oxide and Nitroarene Moieties

The bifunctional hypoxia-specific cytotoxin RB90745, has a nitroimidazole moiety attached to an imidazo[1,2,-a]quinoxaline mono-N-oxide with a spacer/linking group. The reduction chemistry of the drug was studied by pulse radiolysis using the one electron reductant CO₂^˙-. As N-oxides and nitro compounds react with CO₂^˙- at diffusion controlled rates, initial reaction produced a mixture of the nitro radical (λ_max 410 nm) and the N-oxide radical (λ_max 550 nm) in a few microseconds. Subsequently an intramolecular electron transfer (IET) was observed (k = 1.0 ± 0.25 × 10³ s^-1 at pH 5-9), from the N-oxide to the more electron-affinic nitro group. This was confirmed by the first order decay rate of the radical at 550 nm and formation at 410 nm, which was independent of both the concentration of the parent compound and the radicals. The rates of electron transfer and the decay kinetics of the nitro anion radicals were pH dependent and three different pK_aS could be estimated for the one electron reduced species: 5.6 (nitroimidazole group) and 4.3, and 7.6 (N-oxide function). The radicals react with oxygen with rate constants of 3.1 × 10⁷ and 2.8 × 10⁶ dm³ mol^-1 s^-1 observed at 575 nm and 410 nm respectively. Steady state radiolysis studies indicated four electron stoichiometry for the reduction of the compound.     

Activation by reduction of the resting form of cytochrome c oxidase: Tests of different models and evidence for the involvement of CuB

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2 · 10⁴ M⁻¹ · s⁻¹. In the absence of cyanide, ferrocytochrome a₃ appears at a rate (k_obs) of 0.016 s⁻¹. Ferricytochrome a₃ maintains its 418 nm Soret maximum until reduced. The rate of a₃ reduction is independent of dithionite concentration over a range 0.9 mM–131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a₃/cyanide complex occurs at the same rate as a₃ reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a₃ with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a₃ shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a₃. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (k_obs ≈ 0.02 s) entry of a third electron. Above +200 mV, the number of electrons taken up in the initial fast phase drops as a redox center (presumably Cu_A) titrates with an apparent mid-point potential of +240 mV. The slow phase of reduction remains at the more positive redox values. (4) The results are interpreted in terms of an initial fast reduction of cytochrome a (and Cu_A at redox values more negative than +240 mV) followed by a slow reduction of Cu_B. Cu_B reduction is proposed to spin-uncouple cytochrome a₃ to form a cyanide sensitive center, and trigger a conformational change to an activated form of the enzyme with faster intramolecular electron transfer.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Abstract Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a ₁ c ₁ is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c -550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a ₁ c ₁ and aa ₃-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c -550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP⁺ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi . The electron transfer against redox potential from NO₂⁻ to cythochrome c could be pushed through prompt removal by cytochrome aa ₃ of H⁺ formed by the dehydrogenation of NO₂⁻+ H₂O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO₂⁻, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a ₁ c ₁ with NO₂⁻ in vivo.     

Mechanism of O2 generation in reduction and oxidation cycle of ubiquinones in a model of mitochondrial electron transport systems

O⁻₂ generation in mitochondrial electron transport systems, especially the NADPH-coenzyme Q₁₀ oxidoreductase system, was examined using a model system, NADPH-coenzyme Q₁-NADPH-dependent cytochrome P-450 reductase. One electron reduction of coenzyme Q₁ produces coenzyme Q₁ and O⁻₂ during enzyme-catalyzed reduction and O₂ + coenzyme Q₁ are in equilibrium with O⁻₂ + coenzyme Q₁ in the presence of enough O₂. The coenzyme Q₁ produced can be completely eliminated by superoxide dismutase, identical to bound coenzyme Q₁₀ radical produced in a succinate/fumarate couple-KCN-submitochondrial system in the presence of O₂. Superoxide dismutase promotes electron transfer from reduced enzyme to coenzyme Q₁ by the rapid dismutation of O₂⁻ generated, thereby preventing the reduction of coenzyme Q₁ by O⁻₂. The enzymatic reduction of coenzyme Q₁ to coenzyme Q₁H₂ via coenzyme Q₁ is smoothly achieved under anaerobic conditions. The rate of coenzyme Q₁H₂ autoxidation is extremely slow, i.e., second-order constant for [O₂][coenzyme Q₁H₂] = 1.5 M⁻¹ · s⁻¹ at 258 μM O₂, pH 7.5 and 25°C.     

Pyrophosphate import and synthesis by plant mitochondria

The matrix level of pyrophosphate (PPi) in mitochondria isolated from etiolated pea ( Pisum sativum L. cv. Alaska) stems was evaluated, on the basis of an enzymatic assay, to be approx. 0.2 m M . Pyrophosphate could enter from the cytoplasm to the mitochondria via adenine nucleotide translocase (ANT), because F^– and Ca²⁺ (two penetrating PPiase inhibitors) and atractylate (ANT inhibitor) inhibited PPiase activity in isolated mitochondria supplied with PPi. This result was also confirmed by measuring oxygen consumption and membrane potential (ΔΨ) in succinate-energized mitochondria. In a medium free of phosphate (Pi), the addition of PPi before the substrate rendered possible an ADP-stimulated oxygen consumption that was inhibited by F^– or Ca²⁺. In a similar experiment, ADP induced the dissipation of ΔΨ when it was added after the succinate-generated ΔΨ had reached a steady state and, again, F^– inhibited this dissipation. These results imply that PPi enters the mitochondria where it is hydrolyzed to 2 Pi which become available for the H⁺-ATPase (EC 3.6.1.34). In addition, PPi may be synthesized by the H⁺-PPiase (EC 3.6.1.1), acting as a synthase. This evidence arises from the observation that Pi stimulated an oxygen consumption (respiratory control ratio of 1.7) that was inhibited by F^– or Ca²⁺. The physiological role of the mitochondrial H⁺-PPiase is discussed in the light of the consideration that this enzyme can catalyse a readily reversible reaction.     

Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyrⁿ-(X)₃-Lysⁿ⁺⁴) and aldo/keto reductases (AKRs) (Tyrⁿ-(X)₂₈-Lysⁿ⁺²⁹). Tyr⁵¹, Lys⁵⁵ and Lys⁸⁰ of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr⁵¹→ Phe and Lys⁸⁰→Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of -xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues. Replacing Lys⁵⁵ by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr⁵¹ to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.