Near-Infrared Activated Cyanine Dyes As Agents for Photothermal Therapy and Diagnosis of Tumors.

Today, it has become apparent that innovative treatment methods, including those involving simultaneous diagnosis and therapy, are particularly in demand in modern cancer medicine. The development of nanomedicine offers new ways of increasing the therapeutic index and minimizing side effects. The development of photoactivatable dyes that are effectively absorbed in the first transparency window of biological tissues (700-900 nm) and are capable of fluorescence and heat generation has led to the emergence of phototheranostics, an approach that combines the bioimaging of deep tumors and metastases and their photothermal treatment. The creation of near-infrared (NIR) light-activated agents for sensitive fluorescence bioimaging and phototherapy is a priority in phototheranostics, because the excitation of drugs and/or diagnostic substances in the near-infrared region exhibits advantages such as deep penetration into tissues and a weak baseline level of autofluorescence. In this review, we focus on NIR-excited dyes and discuss prospects for their application in photothermal therapy and the diagnosis of cancer. Particular attention is focused on the consideration of new multifunctional nanoplatforms for phototheranostics which allow one to achieve a synergistic effect in combinatorial photothermal, photodynamic, and/or chemotherapy, with simultaneous fluorescence, acoustic, and/or magnetic resonance imaging.

Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c. Optical multichannel detection.

Intramolecular electron transfer in the electrostatic cytochrome c oxidase/cytochrome c complex was investigated using a novel photoactivatable dye. Laser photolysis of thiouredopyrenetrisulfonate (TUPS), covalently linked to cysteine 102 on yeast iso-1-cytochrome c, generates a triplet state of the dye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome c oxidase. Time-resolved optical absorption difference spectra were collected at delay times from 100 ns to 200 ms between 325 and 650 nm. On the basis of singular value decomposition (SVD) and multiexponential fitting, three apparent lifetimes were resolved. A sequential kinetic mechanism is proposed from which the microscopic rate constants and spectra of the intermediates were determined. The triplet state of TUPS donates an electron to cytochrome c with a forward rate constant of approximately 2.0 x 10(4) s(-1). A significant fraction of the triplet returns back to the ground state on a similar time scale. The reduction of cytochrome c is followed by faster electron transfer from cytochrome c to Cu(A), with the equilibrium favoring the reduced cytochrome c. Subsequently, Cu(A) equilibrates with heme a with an apparent rate constant of approximately 1 x 10(4) s(-1). On a millisecond time scale, the oxidized TUPS returns to the ground state and heme a becomes reoxidized. The extracted intermediate spectra are in excellent agreement with model spectra of the postulated intermediates, supporting the proposed mechanism.

Biomaterial engineered electrodes for bioelectronics.

A series of single-cysteine-containing cytochrome c, Cyt c, heme proteins including the wild-type Cyt c (from Saccharomyces cerevisiae) and the mutants (V33C, Q21C, R18C, G1C, K9C and K4C) exhibit direct electrical contact with Au-electrodes upon covalent attachment to a maleimide monolayer associated with the electrode. With the G1C-Cyt c mutant, which includes the cysteine residue in the polypeptide chain at position 1, the potential-induced switchable control of the interfacial electron transfer was observed. This heme protein includes a positively charged protein periphery that surrounds the attachment site and faces the electrode surface. Biasing of the electrode at a negative potential (-0.3 V vs. SCE) attracts the reduced Fe(II)-Cyt c heme protein to the electrode surface. Upon the application of a double-potential-step chronoamperometric signal onto the electrode, where the electrode potential is switched to +0.3 V and back to -0.3 V, the kinetics of the transient cathodic current, corresponding to the re-reduction of the Fe(III)-Cyt c, is controlled by the time interval between the oxidative and reductive potential steps. While a short time interval results in a rapid interfacial electron-transfer, ket1 = 20 s-1, long time intervals lead to a slow interfacial electron transfer to the Fe(III)-Cyt c, ket2 = 1.5 s-1. The fast interfacial electron-transfer rate-constant is attributed to the reduction of the surface-attracted Fe(III)-Cyt c. The slow interfacial electron-transfer rate constant is attributed to the electrostatic repulsion of the positively charged Cyt c from the electrode surface, resulting in long-range electron transfer exhibiting a lower rate constant. At intermediate time intervals between the oxidative and reductive steps, two populations of Cyt c, consisting of surface-attracted and surface-repelled heme proteins, are observed. Crosslinking of a layered affinity complex between the Cyt c and cytochrome oxidase, COx, on an Au-electrode yields an electrically-contacted, integrated, electrode for the four-electron reduction of O2 to water. Kinetic analysis reveals that the rate-limiting step in the bioelectrocatalytic reduction of O2 by the integrated Cyt c/COx electrode is the primary electron transfer from the electrode support to the Cyt c units.

Arylazido-beta-alanine ADP-ribose, a novel irreversible competitive inhibitor of mitochondrial NADH-ubiquinone reductase.

Arylazido-beta-alanine ADP-ribose, a photoreactive analogue of ADP-ribose, was synthesized. In the dark, arylazido-beta-alanine ADP-ribose acts as a competitive reversible inhibitor of mitochondrial NADH-ubiquinone reductase with a K(i) of 37 microM. Upon photolysis, arylazido-beta-alanine ADP-ribose is converted to a potent irreversible active site-directed inhibitor of the enzyme. Photo-induced inhibition of membrane-bound NADH-ubiquinone reductase by arylazido-beta-alanine ADP-ribose is incomplete and results in a 20-fold reduction of the NADH oxidase and 2.5-fold reduction of the energy-dependent NAD(+) reductase activities. The arylazido-beta-alanine ADP-ribose resistant activities (direct and reverse) of the enzyme are characterized by a two orders of magnitude lower affinity to the corresponding substrates compared to those of the uninhibited NADH-ubiquinone reductase. A different kinetic behavior of the inhibited and native enzyme can be explained by invoking the two catalytically competent nucleotide-binding sites model of NADH-ubiquinone reductase.

Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives.

A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction.

Comparison of energization of complex I in membrane particles from Paracoccus denitrificans and bovine heart mitochondria.

The results of preliminary studies of the effects of energization on the catalytic and EPR properties of complex I in tightly coupled membrane vesicles of Paracoccus denitrificans (SPP) are presented. They are compared to those observed in submitochondrial particles from bovine heart (SMP). All signs of energization of complex I detected by EPR in SMP (uncoupler-sensitive splitting of the gz lines of the clusters 2 and a broadening of their gxy lines, a fast-relaxing, piericidin-sensitive ubiquinone-radical signal, and a broad signal around g = 1.94) were also observed with the bacterial enzyme. There were some prominent differences, though. The signal of the fast-relaxing radicals could be evoked both in the presence or absence of reduced clusters 2, suggesting that enhancement of its spin-relaxation rate is caused by coupling to another paramagnet. The signal was hardly affected by the presence of gramicidin. The slow-relaxing radical signal did not disappear upon anaerobiosis, but was detectable for at least another 30 s. The fast-relaxing signal vanished immediately upon anaerobiosis. The activity of the bacterial enzyme during oxidation of NADH by oxygen or reduction of NAD induced by succinate oxidation, was 5-6 times higher than that of the mitochondrial enzyme. Unlike the mitochondrial enzyme, the bacterial enzyme was not inactivated by incubation at 35 degrees C. The spin concentration of the NADH-reducible [2Fe-2S] cluster (1b) was half that of the clusters 2, indicating no difference with the mitochondrial enzyme.

Intramolecular oxidation of cytochrome c by covalently attached sulfoaromatic molecules.

Two photosensitive molecules, 1-maleimidopyrene-3,6,8-trisulfonate (MPTS) and N-acetylaminoethyl-1-aminonaphthalene-5-sulfonate (AEDANS), are employed to drive the intramolecular oxidation of the heme residue in cytochrome c. Intense pulse illumination (60-120 MW cm-2) of MPTS and AEDANS in the aqueous solution by the third harmonic frequency of Nd-Yag laser drives a two successive-photon process of the dyes. The oxidized products originating from the dyes react with variety of electron donors. MPTS and AEDANS residues were covalently linked the Saccharomyces cerevisiae iso-1-cytochrome c by labeling of its single sulfhydryl group. When pulsed by intensive laser beam the heme of the labeled ferrocytochrome c undergoes fast oxidation. Transient absorption spectroscopy was used to directly measure the rate constants for the photoinduced electron-transfer reaction from the ferros heme group to the oxidized dyes. The rate constant was found to be (3.6 +/- 0.4) x 10(4) s-1 for MPTS derivative. The rate of the heme oxidation in AEDANS derivative was faster than response time of our detection system (20 ns). Rapid photooxidation of cytochrome c makes it a useful tool for fast initiation of electron transfer in oxidized direction within complexes of cytochrome c with the other redox proteins.

The iron-sulfur clusters 2 and ubisemiquinone radicals of NADH:ubiquinone oxidoreductase are involved in energy coupling in submitochondrial particles.

The behavior of ubisemiquinone radicals and the iron-sulfur clusters 2 of NADH:ubiquinone oxidoreductase (Complex I) in coupled and uncoupled submitochondrial particles (SMP), oxidizing either NADH or succinate under steady-state conditions, was studied. Multifrequency EPR spectra revealed that the two new g2 lines of the clusters 2, only observed during coupled electron transfer under conditions where energy dissipation is rate-limiting [De Jong, A. M. Ph., Kotlyar, A. B., & Albracht, S. P. J. (1994) Biochim. Biophys. Acta 1186, 163-171], are the result of a spin-spin interaction of 2.8 mT. Investigation of the radical signals present in coupled SMP indicated that more than 90% of the radicals can be ascribed to two types of semiquinones which are bound to Complex I (QI-radicals) or ubiquinol:cytochrome c oxidoreductase (Complex III; QIII-radicals). The presence of QIII-radicals, but not that of QI-radicals, was completely abolished by uncoupler. Part of the QI-radicals weakly interact with the clusters 2 of Complex I. This uncoupler-sensitive interaction can amount to a splitting of the radical EPR signal of at most 1 mT, considerably weaker than the 2.8 mT splitting of the g2 lines of the clusters 2. We propose that the 2.8 mT splitting of these g2 lines results from an energy-induced spin-spin interaction between the two clusters 2 within the TYKY subunit of Complex I. The two clusters 2 show no interaction during electron transfer is uncoupled SMP or in fully-reduced anaerobic-coupled SMP. The results point to a direct role of the Fe-S clusters 2 and the QI-radicals in the mechanism of coupled electron transfer catalyzed by Complex I.

Use of thiouredopyrenetrisulfonate photochemistry for driving electron transfer reactions in aqueous solutions.

Photoexcitation of 1-thiouredopyrene-3,6,8-trisulfonic adducts (TUPS) of amino acids by the third harmonic frequency of a Nd:YAG laser (355 nm) generates the triplet state of the dye with high quantum efficiency. Relaxation of the triplet proceeds in anaerobiosis with a half decay time of 0.5 ms. The relaxation rate increases 100-fold in the presence of dioxygen. A radiative transition between the triplet and the ground state of the dye results in phosphorescent emission centered at 658 nm. The excited state of TUPS, being a strong reductant, can donate its electron to a variety of acceptors. Transient absorption spectroscopy was used to directly measure the photoinduced electron transfer from the excited dye to rhodamine B (RB) and cytochrome c. The reaction with RB was followed by monitoring the oxidation of the triplet state of TUPS at 487 nm (epsilon = 25 000 +/- 5 000 M-1 cm-1) or the reduction of RB at 553 nm. The second order rate constant for the reaction was found to be (2.5 +/- 0.2) x 10(9) M-1 s-1, a value compatible with that for diffusion controlled reactions. When directed to cytochrome c the photoinduced perturbation causes rapid reduction of the protein's heme group, seen as a monophasic increase of absorbance at 550 nm. The combination of appropriate redox properties with the capability of covalent protein modification makes the dye useful for initiation and analysis of electron transfer reactions in chemical and biological systems.

Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate cytochrome c derivatives.

A novel method for initiation of intramolecular electron transfer reactions in cytochrome c is reported. The method is based on photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS) by the third harmonic frequency of a Nd:YAG laser (355 nm), the reaction that generates the low-potential triplet state of the dye with high quantum efficiency. TUPS derivatives of horse heart cytochrome c singly labeled at specific lysine residues were prepared and purified to homogeneity by ion-exchange high-pressure liquid chromatography. Eight derivatives were characterized by determination of the location of the modification, reduction potentials, and measurement of enzymatic activity with cytochrome oxidase. Transient absorption spectroscopy was used to directly measure the rate constants for the electron transfer reaction from the photoexcited triplet state of TUPS to the oxidized heme group and the back reaction from the ferrous heme to the oxidized dye. For all singly labeled derivatives, the rate constants for heme reduction were 1 or 2 orders of magnitude larger than for its reoxidation, consistent with the greater thermodynamic driving force for the oxidation reaction. Analysis of the variation of electron-transfer rates with the distance separating the dye and the heme reveals a value of coupling decay constant (beta) of 0.46 A-1. Rapid and effective photoreduction of cytochrome c makes it a useful tool for fast initiation of electron transfer in the reductive direction within complexes of cytochrome c with other redox proteins.

Energy-induced structural changes in NADH:Q oxidoreductase of the mitochondrial respiratory chain.

The reaction of coupled submitochondrial particles (SMP) with NADH was studied in the absence and presence of the uncoupler gramicidin, both in pre-steady-state and steady-state experiments. It was shown that the formation of ubisemiquinones associated with NADH:Q oxidoreductase is insensitive to uncouplers. It was found, however, that in the absence of gramicidin the ubisemiquinone showed a noticeably faster relaxation than in the presence of this uncoupler. During steady-state oxidation of NADH by coupled submitochondrial particles, the EPR signal of iron-sulphur cluster 2 of complex I, the cluster that is generally believed to be the electron donor for ubiquinone, showed some remarkable changes. Its gz line seemed to disappear from the spectrum, although the gxy line remained clearly present. Detailed EPR analysis indicated that (a component of) the gz line shifted to higher field. The temperature dependence of the EPR signal of cluster 2 was affected as well. In the presence of uncoupler the EPR properties of cluster 2 were indistinguishable from those in particles that showed no intrinsic coupling. These experiments strongly indicate that the coordination of cluster 2 is different in energized and non-energized SMP. The pre-steady-state reaction between these submitochondrial particles and NADH showed that the uncoupler-sensitive changes in both the ubisemiquinone and cluster 2 became effective between 9 ms and 30 ms. Similar changes were observed during succinate-driven reverse electron transfer. This report shows, for the first time, energy-induced structural changes in NADH:Q oxidoreductase.

The dynamics of proton transfer at the C side of the mitochondrial membrane: picosecond and microsecond measurements.

The excited-state proton emitter, pyranine (8-hydroxypyrene-1,3,6-trisulfonate), was introduced into the inner aqueous space of inside-out submitochondrial particles (SMP). Upon initiation of respiration, the dye recorded acidification of this space. Incorporation of high concentrations of the dye (approximately 100 nmol/mg of protein) had no effect on the respiratory functions of the vesicles, nor on their capacity to execute delta microH(+)-coupled reverse electron transfer. The respiratory control ratio (RCR) remained as high as RCR > 4. Pulse irradiation of the dye caused photodissociation of the proton from the 8-hydroxy position. The release of the proton and its reaction with the matrix of the inner space of SMP were monitored at two time intervals: nanosecond fluorimetry measured the dissociation of the proton from the excited dye molecule (phi OH*), while microsecond spectroscopy followed the reaction between the proton and the ground-state anion (phi O-). Numerical integration of the differential rate equations describes the diffusion of protons in the perturbed system. The nanosecond measurements yield the physical characteristics of the aqueous phase that dissolves the dye. The apparent dielectric constant of that space is rather low (epsilon = 20). The diffusion coefficient of the proton is 2.3 x 10(-5) cm2/s, and the activity of water is aH2O = 0.87. All of these values imply that a large fraction of the intervesicular aqueous phase is taken up by the hydration layer of the lipids and proteins of the C side of the membrane. The microsecond dynamics measurements indicate that the rates of proton binding to the membrane surface components reach an equilibrium within 60 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of bulk protons with a mitochondrial inner membrane preparation: time-resolved measurements and their analysis.

The laser-induced proton pulse technique [Gutman, M. (1986) Methods Enzymol. 127, 522-538] was applied on suspensions of submitochondrial vesicles, and the exchange of protons between the bulk and the mitochondrial membranes was measured in the time-resolved domain with a submicrosecond resolution. The protons were discharged by photoexcitation of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) by a short laser pulse, and the reprotonation of the pyranine anion was monitored at 457.8 nm. In parallel, the protonation of the membrane was followed at 496.5 nm, looking at the transient absorbance of fluorescein, covalently attached to the M side of the membrane. The analysis of the relaxation dynamics was carried out by a simulation procedure that reconstructs the observed dynamics of the two chromophores. The analysis revealed the presence of the membrane indigenous buffering moieties. The low-pK buffer (pK 4.1) was present in a quantity of 100 +/- 20 nmol/mg of protein, and its kinetics indicate that it appears in multianionic clusters bearing a negative electric charge. The medium-pK buffer (pK 6.9) was present in a larger quantity (200 +/- 20 nmol/mg), and its kinetic parameter indicated clustering into positively charged domains. Both types of indigenous buffer reacted with the proton and pyranine anion in unhindered diffusion-controlled reactions. On the other hand, the exchange of protons between the indigenous buffer moieties was rather slow. No evidence was found for the presence of sites capable of retaining a proton, secluded from the bulk, for a time frame longer than 100 microseconds as required by the models of localized proton gradient.

The effect of delta mu H+ on the interaction of rotenone with complex I of submitochondrial particles.

The inhibition by rotenone of the forward (NADH-oxidase) and reverse (delta mu H(+)-dependent succinate-NAD+ reductase activities of submitochondrial vesicles was measured. The inhibition of NADH-oxidase, measured in the presence of uncoupler, followed a monophasic inhibition curve with Ki < or = 2 nM. The reverse electron flow was only partially (40%) inhibited at these rotenone concentrations. The rest of the activity was less sensitive to the inhibitor (Ki approximately 30 nM). The lower affinity for the inhibitor of the reverse electron flow is a consequence of enhanced rate of rotenone dissociation caused by the high delta mu H+ value required for this reaction. The analysis of the results indicates that the AS-SMP preparation consists of two subpopulations: one with a relatively low degree of coupling, which exhibits high sensitivity to rotenone and the other which is highly coupled with lower affinity to the inhibitor.

Interaction of ubiquinone and vitamin K3 with mitochondrial succinate-ubiquinone oxidoreductase.

Two new methods have been devised for measuring fumarate reduction by beef heart succinate-ubiquinone oxidoreductase with quinols as electron donors. In one assay the quinone is maintained in the reduced state by coupling fumarate reduction with the DT-diaphorase reaction, in the other assay by the presence of excess dithionite. The advantages of these methods are discussed, along with preliminary characterization of the quinol-protein interaction.

Effect of Ca2+ ions on the slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase.

Slow active/inactive transition of the membrane-bound mitochondrial NADH-ubiquinone reductase (Kotlyar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 1019, 151-158) is sensitive to Ca2+ and other divalent cations. Millimolar concentrations of Ca2+ drastically reduce the rate of the turnover-dependent activation of NADH-ubiquinone reductase. When NADH oxidase, the rotenone-sensitive NADH-ubiquinone reductase or the succinate-supported delta mu H+-dependent NAD+ reduction were initiated by the deactivated enzyme preparations all the three activities were strongly inhibited by Ca2+; no sensitivity of these reactions to Ca2+ was observed when the assays were started by the activated enzyme preparations. The affinity of the deactivated enzyme to polyvalent cations was in the following order: Ni2+ greater than Co2+ greater than La3+ greater than Mn2+ greater than Ca2+ approximately Mg2+ greater than Ba2+. Monovalent metal cations had no effect on the slow turnover-dependent enzyme activation. The apparent affinity of the deactivated enzyme to Ca2+ was strongly pH-dependent. The KCa2+ values of 5.7 mM and 0.6 mM at pH 7.5 and 8.5 were determined from the presteady-state kinetics parameters. The spontaneous temperature-dependent deactivation of the enzyme was insensitive to Ca2+. Ca2+ increases the reactivity of the enzyme sulfhydryl group in the deactivated preparations towards N-ethylmaleimide. This effect was also used to quantitate Ca2+ affinity for the enzyme. The KCa2+ values of 1.2 mM and 0.4 mM at pH 8.0 and 9.0, respectively, were determined. The data obtained suggest that Ca2+ content in the mitochondrial matrix may play an important role in the control of NADH oxidation by the respiratory chain.

Slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase.

NADH-ubiquinone reductase of bovine heart submitochondrial particles as prepared is unable to catalyze either the direct or reverse electron transfer from NADH to ubiquinone. The deactivated state of the enzyme in coupled particles was revealed as: (i) the absence of the rotenone-sensitive, delta mu H(+)-dependent succinate-ferricyanide reductase activity; (ii) a prominent lag in the aerobic succinate-supported, delta mu H(+)-dependent NAD+ reduction; and (iii) a lag in the rotenone-sensitive NADH-ubiquinone reductase or NADH oxidase activities. Being inactive as NADH-ubiquinone reductase (direct or reverse), the enzyme is fully active as rotenone-insensitive NADH-ferricyanide reductase. The enzyme can be activated by preincubation with substrates (NADH or NADPH) only under the conditions where the turnover of the NADH-ubiquinone reductase reaction (but not in the NADH-ferricyanide reductase) occurs. Partial activation of the enzyme was observed when the particles were preincubated with rotenone. Neither NADH under the conditions when the ubiquinone pool was reduced nor succinate plus delta mu H+ or dithionite were able to activate the enzyme. Once activated, the enzyme remains in the active state for quite a long time (more than 5 h at 0 degree C). The deactivation rate is extremely temperature-dependent, being insensitive to NAD+, ferricyanide or succinate. A comparison of the enzyme activation/deactivation kinetics showed that the same mechanism is involved in the slow activation of the direct and reverse electron transfer from NADH to ubiquinone. Activated particles catalyze the aerobic delta mu H(+)-dependent succinate-supported reverse electron transfer in the absence of ATP at a rate comparable with that of NADH-ubiquinone reductase.

Coupling site I and the rotenone-sensitive ubisemiquinone in tightly coupled submitochondrial particles.

The rotenone-sensitive g = 2.00 low temperature EPR signal attributed to ubisemiquinone is observed in submitochondrial particles during coupled electron transfer from NADH to oxygen and from succinate to NAD+. The signal is seen only in the presence of oligomycin added to induce the respiratory control (7-9 with NADH and 3-4 with succinate) and it disappears in the presence of uncouplers (CCCP or gramicidin D). No reduction of the iron-sulfur center N-2 in the presence of 20 mM succinate and cyanide is observed, thus suggesting that N-2 is not in equilibrium with the ubiquinone pool. A hypothesis is proposed on delta mu H+ generation coupled with electron transfer between iron-sulfur center N-2 and the ubiquinone pool.

Identification of the high-molecular-mass mitochondrial oxaloacetate keto-enol tautomerase as inactive aconitase.

The homogeneous bovine heart mitochondrial high-molecular-mass oxaloacetate keto-enol tautomerase [(1988) Biochim. Biophys. Acta 936, 10-19] is shown to be an iron-sulfur protein as revealed by the enzyme spectral properties and direct chemical determination of non-heme iron and acid-labile sulfur. The protein is capable of catalysing the aconitase reaction after treatment with ferrous ions under anaerobic conditions. Treatment of the 'activated' protein with N-ethylmaleimide results in the simultaneous irreversible loss of the oxaloacetate keto-enol tautomerase and aconitase activities. The effects of some substrates and inhibitors on both activities show that the same catalytic site is involved in the oxaloacetate tautomerase and aconitase reactions. It is concluded that the protein previously described as a 80 kDa oxaloacetate keto-enol tautomerase is inactive aconitase.

Regulation of succinate dehydrogenase and tautomerization of oxaloacetate.

Highly purified succinate-ubiquinone reductase catalyzes the oxidation of L- or D-malate with a Km and initial Vmax equal to approximately 10(-3) M and approximately 100 nmol/min/mg of protein, respectively. The malate dehydrogenase activity of succinate dehydrogenase rapidly decreases regardless of the presence of glutamate plus glutamate-oxaloacetate transaminase. The inhibitor trapping system, however, prevents the inactivation of succinate dehydrogenase under the conditions when the rate of tautomeric oxaloacetate enol in equilibrium oxaloacetate ketone interconversion is high. These results suggest that enol oxaloacetate is an immediate product of malate oxidation at the succinate dehydrogenase active site. Two proteins (Mr 37 and 80 kD) which catalyze the oxaloacetate tautomerase reaction were isolated from the mitochondrial matrix. Some physico-chemical and kinetic properties of these enzymes were characterized. The larger protein was identified as inactive aconitase. The system containing succinate dehydrogenase, L-malate, glutamate plus transaminase and oxaloacetate tautomerase was reconstituted. Such a system is capable of oxidizing malate to aspartate without rapid inactivation of succinate dehydrogenase. Taken together, the data obtained emphasize a significant role of enzymatic oxaloacetate tautomerization in the control of the succinate dehydrogenase activity in the mitochondrial matrix.