Vibrational modes of ubiquinone in cytochrome bo(3) from Escherichia coli identified by Fourier transform infrared difference spectroscopy and specific (13)C labeling.

In this study we present the infrared spectroscopic characterization of the bound ubiquinone in cytochrome bo(3) from Escherichia coli. Electrochemically induced Fourier transform infrared (FTIR) difference spectra of DeltaUbiA (an oxidase devoid of bound ubiquinone) and DeltaUbiA reconstituted with ubiquinone 2 and with isotopically labeled ubiquinone 2, where (13)C was introduced either at the 1- or at the 4-position of the ring (C=O groups), have been obtained. The vibrational modes of the quinone bound to the discussed high-affinity binding site (Q(H)) are compared to those from the synthetic quinones in solution, leading to the assignment of the C=O modes to a split signal at 1658/1668 cm(-)(1), with both carbonyls similarly contributing. The FTIR spectra of DeltaUbiA reconstituted with the labeled quinones indicate an essentially symmetrical and weak hydrogen bonding of the two C=O groups from the neutral quinone with the protein and distinct conformations of the 2- and 3-methoxy groups. Perturbations of the vibrational modes of the 5-methyl side groups are discussed for a signal at 1452 cm(-)(1). Only negligible shifts of the aromatic ring modes can be reported for the reduced and the protonated form of the quinone. Alterations of the protein upon quinone binding are reflected in the electrochemically induced FTIR difference spectra. In particular, difference signals at 1640-1633 cm(-)(1) and 1700-1670 cm(-)(1) indicate variations of beta-sheet secondary structure elements and loops, bands at 1706 and 1678 cm(-)(1) are tentatively attributed to individual amino acids, and a difference signal a 1540 cm(-)(1) is discussed to reflect an influence on C=C modes of the porphyrin ring or on deprotonated propionate groups of the hemes. Further tentative assignments are presented and discussed. The (13)C labeling experiments allow the assignment of the vibrational modes of a bound ubiquinone 8 in the electrochemically induced FTIR difference spectra of wild-type bo(3).     

An isotope-edited FTIR investigation of the role of Ser-L223 in binding quinone (QB) and semiquinone (QB-) in the reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (Q(B)) site. Several nearby residues are important for both binding and redox chemistry involved in the light-induced conversion from Q(B) to quinol Q(B)H(2). Ser-L223 is one of the functionally important residues located near Q(B). To obtain information on the interaction between Ser-L223 and Q(B) and Q(B)(-), isotope-edited Q(B)(-)/Q(B) FTIR difference spectra were measured in a mutant RC in which Ser-L223 is replaced with Ala and compared to the native RC. The isotope-edited IR fingerprint spectra for the C=O [see text] and C=C [see text] modes of Q(B) (Q(B)(-)) in the mutant are essentially the same as those of the native RC. These findings indicate that highly equivalent interactions of Q(B) and Q(B)(-) with the protein occur in both native and mutant RCs. The simplest explanation of these results is that Ser-L223 is not hydrogen bonded to Q(B) or Q(B)(-) but presumably forms a hydrogen bond to a nearby acid group, preferentially Asp-L213. The rotation of the Ser OH proton from Asp-L213 to Q(B)(-) is expected to be an important step in the proton transfer to the reduced quinone. In addition, the reduced quinone remains firmly bound, indicating that other distinct hydrogen bonds are more important for stabilizing Q(B)(-). Implications on the design features of the Q(B) binding site are discussed.     

Architecture of the Qo site of the cytochrome bc1 complex probed by superoxide production

Although several X-ray structures have been determined for the mitochondrial cytochrome (cyt) bc(1) complex, none yet shows the position of the substrate, ubiquinol, in the quinol oxidase (Q(o)) site. In this study, the interaction of molecular oxygen with the reactive intermediate Q(o) semiquinone is used to probe the Q(o) site. It has been known for some time that partial turnover of the cyt bc(1) complex in the presence of antimycin A, a Q(i) site inhibitor, results in accumulation of a semiquinone at the Q(o) site, which can reduce O(2) to superoxide (O(2)(*)(-)). It was more recently shown that myxothiazol, which binds close to the cyt b(L) heme in the proximal Q(o) niche, also induces O(2)(*)(-) production. In this work, it is shown that, in addition to myxothiazol, a number of other proximal Q(o) inhibitors [including (E)-beta-methoxyacrylate-stilbene, mucidin, and famoxadone] also induce O(2)(*)(-) production in the isolated yeast cyt bc(1) complex, at approximately 50% of the V(max) observed in the presence of antimycin A. It is proposed that proximal Q(o) site inhibitors induce O(2)(*)(-) production because they allow formation, but not oxidation, of the semiquinone at the distal niche of the Q(o) site pocket. The apparent K(m) for ubiquinol at the Q(o) site in the presence of proximal Q(o) site inhibitors suggests that the "distal niche" of the Q(o) pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin and ubiquinol can bind simultaneously to the Q(o) site with virtually no steric hindrance, whereas progressively bulkier inhibitors exhibit increasing overlap. The fact that partial turnover of the Q(o) site is possible even with bound proximal Q(o) site inhibitors is consistent with the participation of two separate functional Q(o) binding niches, occupied simultaneously or sequentially.     

-deltaG(AB) and pH dependence of the electron transfer from P(+)Q(A)(-)Q(B) toP(+)Q(A)Q(B)(-) in Rhodobacter sphaeroides reaction centers

The electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) can occur in two phases with a well-characterized 100 micros component (tau(2)) and a faster process occurring in less than 10 micros (tau(1)). The fast reaction is clearly seen when the native ubiquinone-10 at Q(A) is replaced with naphthoquinones. The dependence of tau(1) on the free-energy difference between the P(+)Q(A)(-)Q(B) and P(+)Q(A)Q(B)(-) states (-) and on the pH was measured using naphthoquinones with different electrochemical midpoint potentials as Q(A) in Rhodobacter sphaeroides reaction centers (RCs) and in RCs where - is changed by mutation of M265 in the Q(A) site from Ile to Thr (M265IT). Q(B) was ubiquinone (UQ(B)) in all cases. Electron transfer was measured by using the absorption differences of the naphthosemiquinone at Q(A) and the ubisemiquinone at Q(B) between 390 and 500 nm. As - was changed from -90 to -250 meV tau(1) decreased from 29 to 0.2 micros. The free-energy dependence of tau(1) provides a reorganization energy of 850 +/- 100 meV for the electron transfer from Q(A)(-) to Q(B). The slower reaction at tau(2) is free-energy independent, so processes other than electron transfer determine the observed rate. The fraction of the reaction at tau(1) increases with increasing driving force and is 100% of the reaction when - is approximately 100 meV more favorable than in the native RCs with ubiquinone as Q(A). The fast phase, tau(1), is pH independent from pH 6 to 11 while tau(2) slows above pH 9. As the Q(A) isoprene tail length is increased from 2 to 10 isoprene units the fraction at tau(1) decreases. However, tau(1), tau(2), and the fraction of the reaction in each phase are independent of the tail length of UQ(B).     

Ras regulation by reactive oxygen and nitrogen species

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. We have previously shown that both NO/O(2) (via nitrogen dioxide, (*)NO(2)) and superoxide radical anion (O(2)(*)(-)) promote Ras guanine nucleotide dissociation. We now show that hydrogen peroxide in the presence of transition metals (i.e., H(2)O(2)/transition metals) and peroxynitrite also trigger radical-based Ras guanine nucleotide dissociation. The primary redox-active reaction species derived from H(2)O(2)/transition metals and peroxynitrite is O(2)(*)(-) and (*)NO(2), respectively. A small fraction of hydroxyl radical (OH(*)) is also present in both. We also show that both carbonate radical (CO(3)(*)(-)) and (*)NO(2), derived from the mixture of peroxynitrite and bicarbonate, facilitate Ras guanine nucleotide dissociation. We further demonstrate that NO/O(2) and O(2)(*)(-) promote Ras GDP exchange with GTP in the presence of a radical-quenching agent, ascorbate, or NO, and generation of Ras-GTP promotes high-affinity binding of the Ras-binding domain of Raf-1, a downstream effector of Ras. S-Nitrosylated Ras (Ras-SNO) can be formed when NO serves as a radical-quenching agent, and hydroxyl radical but not (*)NO(2) or O(2)(*)(-) can further react with Ras-SNO to modulate Ras activity in vitro. However, given the lack of redox specificity associated with the high redox potential of OH(*), it is unclear whether this reaction occurs under physiological conditions.     

Primary quinone (QA) binding site of bacterial photosynthetic reaction centers: mutations at residue M265 probed by FTIR spectroscopy

In the primary quinone (Q(A)) binding site of Rb. sphaeroides reaction centers (RCs), isoleucine M265 is in extensive van der Waals contact with the ubiquinone headgroup. Substitution of threonine or serine for this residue (mutants M265IT and M265IS), but not valine (mutant M265IV), lowers the redox midpoint potential of Q(A) by about 100 mV (Takahashi et al. (2001) Biochemistry 40, 1020-1028). The unexpectedly large effect of the polar substitutions is not due to reorientation of the methoxy groups as similar redox potential changes are seen for these mutants with either ubiquinone or anthraquinone as Q(A). Using FTIR spectroscopy to compare Q(A)(-)/Q(A) IR difference spectra for wild type and the M265 mutant RCs, we found changes in the polar mutants (M265IT and M265IS) in the quinone C[double bond]O and C[double bond]C stretching region (1600-1660 cm(-1)) and in the semiquinone anion band (1440-1490 cm(-1)), as well as in protein modes. Modeling the mutations into the X-ray structure of the wild-type RC indicates that the hydroxyl group of the mutant polar residues, Thr and Ser, is hydrogen bonded to the peptide C[double bond]O of Thr(M261). It is suggested that the mutational effect is exerted through the extended backbone region that includes Ala(M260), the hydrogen bonding partner to the C1 carbonyl of the quinone headgroup. The resulting structural perturbations are likely to include lengthening of the hydrogen bond between the quinone C1[double bond]O and the peptide NH of Ala(M260). Possible origins of the IR spectroscopic and redox potential effects are discussed.     

Effect of dicumarol,a Nad(P)h: quinone acceptor oxidoreductase 1 (DT-diaphorase) inhibitor on ubiquinone redox cycling in cultured rat hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 μM dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro , was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 μM dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Photoreduction of the quinone pool in the bacterial photosynthetic membrane: identification of infrared marker bands for quinol formation

The photoreduction of the quinone (Q) pool in the photosynthetic membrane of the purple bacterium Rhodobacter sphaeroides was investigated by steady-state and time-resolved Fourier transform infrared difference spectroscopy. The results are consistent with the existence of a homogeneous Q pool inside the chromatophore membrane, with a size of around 20 Q molecules per reaction center. IR marker bands for the quinone/quinol (Q/QH(2)) redox couple were recognized. QH(2) bands are identified at 1491, 1470, 1433 and 1388-1375 cm(-1). The 1491 cm(-1) band, which is sensitive to (1)H/(2)H exchange, is assigned to a C-C ring mode coupled to a C-OH mode. A feature at approximately 1743/1720 cm(-1) is tentatively related to a perturbation of the carbonyl modes of phospholipid head groups induced by QH(2) formation. Complex conformational changes of the protein in the amide I and II spectral ranges are also apparent during reduction and reoxidation of the Q pool.     

Analysis of microbial community structure in a biofilm on membrane surface in the submerged membrane bioreactor treating domestic wastewater on the basis of respiratory quinone profiles

The objective of this study was to investigate the microbial community structure of the biofouling film formed on hollow-fiber membrane surfaces in the submerged membrane bioreactor (SMBR) with a nitrification-denitrification process. In this experiment, aeration was conducted intermittently (60 min off, 90 min on) cyclic anoxic and oxic conditions in the SMBR. The dominant quinone types of biofilm on the membrane surface in an intermittently aerated SMBR were ubiquinone (UQs)-8, -10, followed by menaquinones (MKs)-8(H4), -8(H2) and -7, but those of suspended microorganisms were UQ-8, UQ-10 followed by MKs-8, -9(H4) and -6. The change in quinone profiles of biofilm on the membrane surface suggested that UQ-9, MK-7, MK-8(H2) and MK-8(H4) contributed to microbiological fouling in the intermittently aerated SMBR treating domestic wastewater. The microbial diversities of suspended microorganisms and biofilm, calculated based on the composition of all quinones, were 9.5 and 10.9, respectively.     

Structural basis for the quinone reduction in the bc1 complex: a comparative analysis of crystal structures of mitochondrial cytochrome bc1 with bound substrate and inhibitors at the Qi site

Cytochrome bc(1) is an integral membrane protein complex essential to cellular respiration and photosynthesis. The Q cycle reaction mechanism of bc(1) postulates a separated quinone reduction (Q(i)) and quinol oxidation (Q(o)) site. In a complete catalytic cycle, a quinone molecule at the Q(i) site receives two electrons from the b(H) heme and two protons from the negative side of the membrane; this process is specifically inhibited by antimycin A and NQNO. The structures of bovine mitochondrial bc(1) in the presence or absence of bound substrate ubiquinone and with either the bound antimycin A(1) or NQNO were determined and refined. A ubiquinone with its first two isoprenoid repeats and an antimycin A(1) were identified in the Q(i) pocket of the substrate and inhibitor bound structures, respectively; the NQNO, on the other hand, was identified in both Q(i) and Q(o) pockets in the inhibitor complex. The two inhibitors occupied different portions of the Q(i) pocket and competed with substrate for binding. In the Q(o) pocket, the NQNO behaves similarly to stigmatellin, inducing an iron-sulfur protein conformational arrest. Extensive binding interactions and conformational adjustments of residues lining the Q(i) pocket provide a structural basis for the high affinity binding of antimycin A and for phenotypes of inhibitor resistance. A two-water-mediated ubiquinone protonation mechanism is proposed involving three Q(i) site residues His(201), Lys(227), and Asp(228).     

Site specific labeling of Rhodobacter sphaeroides reaction centers with dye probes for surface pH measurements

Covalently bound pH sensitive dyes are an important tool for characterizing the proteolytic reactions of protein complexes that play key roles in biological energy transduction. Here we demonstrate the feasibility of this method for photosynthetic reaction centers (RCs) for the first time, by the highly selective attachment of two thiol reactive derivatives of fluorescein to the two H subunit cysteines of the photosynthetic RC from Rhodobacter sphaeroides R-26 The pK(a) shifts of the dyes upon binding to the protein and in response to high salt were measured, and interpreted based on the structure of the RC. 2-[(5-fluoresceinyl)aminocarbonyl]ethyl-methanethiosulfonate was attached to Cys H156 and fluorescein-5-maleimide to Cys H234. By following the absorption changes of bound fluorescein (500 nm), and those of the hydrophilic pH indicator 8-hydroxypyrene-1,3,6-tris-sulfonic acid (468 nm), the surface and bulk pH were monitored separately with less than 5% crosstalk. Flash-induced proton uptake and external calibrations by mixing with aliquots of acid were measured in different redox states of the RCs. The results indicate that the charge in the quinone acceptor complex after flash activation (primary quinone acceptor (Q(A))- or secondary quinone acceptor (Q(B))-) has no effect on the surface pH and potential in the vicinity of these two attachment sites, between pH 6.5 and 9. Application of the method to other surface locations is discussed.     

Effects of copper and zinc ions on photosystem II studied by EPR spectroscopy.

The effect of Zn(2+) or Cu(2+) ions on Mn-depleted photosystem II (PS II) has been investigated using EPR spectroscopy. In Zn(2+)-treated and Cu(2+)-treated PS II, chemical reduction with sodium dithionite gives rise to a signal attributed to the plastosemiquinone, Q(A)(*)(-), the usual interaction with the non-heme iron being lost. The signal was identified by Q-band EPR spectroscopy which partially resolves the typical g-anisotropy of the semiquinone anion radical. Illumination at 200 K of the unreduced samples gives rise to a single organic free radical in Cu(2+)-treated PS II, and this is assigned to a monomeric chlorophyll cation radical, Chl a(*)(+), based on its (1)H-ENDOR spectrum. The Zn(2+)-treated PS II under the same conditions gives rise to two radical signals present in equal amounts and attributed to the Chl a(*)(+) and the Q(A)(*)(-) formed by light-induced charge separation. When the Cu(2+)-treated PS II is reduced by sodium ascorbate, at >/=77 K electron donation eliminates the donor-side radical leaving the Q(A)(*)(-) EPR signal. The data are explained as follows: (1) Cu(2+) and Zn(2+) have similar effects on PS II (although higher concentrations of Zn(2+) are required) causing the displacement of the non-heme Fe(2+). (2) In both cases chlorophyll is the electron donor at 200 K. It is proposed that the lack of a light-induced Q(A)(*)(-) signal in the unreduced Cu(2+)-treated sample is due to Cu(2+) acting as an electron acceptor from Q(A)(*)(-) at low temperature, forming the Cu(+) state and leaving the electron donor radical Chl a(*)(+) detectable by EPR. (3) The Cu(2+) in PS II is chemically reducible by ascorbate prior to illumination, and the metal can therefore no longer act as an electron acceptor; thus Q(A)(*)(-) is generated by illumination in such samples. (4) With dithionite, both the Cu(2+) and the quinone are reduced resulting in the presence of Q(A)(*)(-) in the dark. The suggested high redox potential of Cu(2+) when in the Fe(2+) site in PS II is in contrast to the situation in the bacterial reaction center where it has been shown in earlier work that the Cu(2+) is unreduced by dithionite. It cannot be ruled out however that Q(A)-Cu(2+) is formed and a magnetic interaction is responsible for the lack of the Q(A)(-) signal when no exogenous reductant is present. With this alternative possibility, the effects of reductants would be explained as the loss of Cu(2+) (due to formation of Cu(+)) leading to loss of the Cu(2+) from the Fe(2+) site due to the binding equilibrium. The quite different binding and redox behavior of the metal in the iron site in PS II compared to that of the bacterial reaction center is presumably a further reflection of the differences in the coordination of the iron in the two systems.     

Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal (Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.     

Identification of the proton pathway in bacterial reaction centers: cooperation between Asp-M17 and Asp-L210 facilitates proton transfer to the secondary quinone (QB)

The reaction center (RC) from Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, Q(B) (the secondary quinone electron acceptor), to form quinol, Q(B)H2. Asp-L210 and Asp-M17 have been proposed to be components of the pathway for proton transfer [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. To test the importance of these residues for efficient proton transfer, the rates of the proton-coupled electron-transfer reaction k(AB)(2) (Q(A-*)Q(B-*) + H+ <==>Q(A-*)Q(B)H* --> Q(A)Q(B)H-) and its associated proton uptake were measured in native and mutant RCs, lacking one or both Asp residues. In the double mutant RCs, the k(AB)(2) reaction and its associated proton uptake were approximately 300-fold slower than in native RCs (pH 8). In contrast, single mutant RCs displayed reaction rates that were < or =3-fold slower than native (pH 8). In addition, the rate-limiting step of k(AB)(2) was changed from electron transfer (native and single mutants) to proton transfer (double mutant) as shown from the lack of a dependence of the observed rate on the driving force for electron transfer in the double mutant RCs compared to the native or single mutants. This implies that the rate of the proton-transfer step was reduced (> or =10(3)-fold) upon replacement of both Asp-L210 and Asp-M17 with Asn. Similar, but less drastic, differences were observed for k(AB)(1), which at pH > or =8 is coupled to the protonation of Glu-L212 [(Q(A-*)Q(B))-Glu- + H+ --> (Q(A)Q(B-*)-GluH]. These results show that the pathway for proton transfer from solution to reduced Q(B) involves both Asp-L210 and Asp-M17, which provide parallel branches to the proton-transfer pathway and through their electrostatic interaction have a cooperative effect on the proton-transfer rate. A possible mechanism for the cooperativity is discussed.     

Fourier transform infrared spectrum of the secondary quinone electron acceptor Q(B) in photosystem II

Fourier transform infrared difference spectra upon single reduction of the secondary quinone electron acceptor Q(B) in photosystem II (PSII), without a contribution from the electron donor-side signals, were obtained for the first time using Mn-depleted PSII core complexes of the thermophilic cyanobacterium Thermosynechococcus elongatus. The Q(B)(-)/Q(B) difference spectrum exhibited a strong C...O stretching band of the semiquinone anion at 1480 cm(-)(1), the frequency higher by 2 cm(-)(1) than that of the corresponding band of Q(A)(-), in agreement with the previous S(2)Q(B)(-)/S(1)Q(B) spectrum of the PSII membranes of spinach [Zhang, H., Fischer, G., and Wydrzynski, T. (1998) Biochemistry 37, 5511-5517]. Also, several peaks originating from the Fermi resonance of coupled His modes with its strongly H-bonded NH vibration were observed in the 2900-2600 cm(-)(1) region, where the peak frequencies were higher by 7-24 cm(-)(1) compared with those of the Q(A)(-)/Q(A) spectrum. These frequency differences suggest that H-bond interactions of the CO groups, especially with a His side chain, are different between Q(B)(-) and Q(A)(-). Furthermore, a prominent positive peak was observed at 1745 cm(-)(1) in the C=O stretching region of COOH or ester groups in the Q(B)(-)/Q(B) spectrum. The peak frequency was unaffected by D(2)O substitution, indicating that this peak does not arise from a COOH group but probably from the 10a-ester C=O group of the pheophytin molecule adjacent to Q(B). The absence of protonation of carboxylic amino acids upon Q(B)(-) formation in contrast to the previous observation in the purple bacterium Rhodobacter sphaeroides suggests that the protonation mechanism of Q(B) in PSII is different from that of bacterial reaction centers.     

Superoxide radical-initiated apoptotic signalling pathway in selenite-treated HepG(2) cells: mitochondria serve as the main target

The exact role of superoxide radicals (O(2)(*)(-)) in apoptosis is still a matter of debate. The main objective of the present study is to evaluate the apoptotic signalling pathway initiated by O(2)(*)(-). The reductive reaction of sodium selenite with glutathione was used as the intracellular O(2)(*)(-)-generating system. When cells were exposed to 5 to 25 microM selenite, a temporal pattern of apoptotic events was observed following the elevation of O(2)(*)(-), in which cytochrome c release and mitochondrial depolarization preceded caspase-3 activation and DNA fragmentation. The simultaneous treatment with N-acetylcysteine and 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl markedly reduced O(2)(*)(-) level and suppressed the mitochondrial changes and the downstream apoptotic events. Moreover, pretreatment with cyclosporin A plus trifluoperazine, two mitochondrial permeability transition (MPT) inhibitors, was capable of attenuating O(2)(*)(-)-mediated cytochrome c release and mitochondrial depolarization, and subsequently inhibiting apoptosis. Thus, the present results provide convincing evidence that O(2)(*)(-) generated from the reductive reaction of selenite with GSH is capable of triggering a mitochondria-dependent apoptotic pathway. Such knowledge may not only help to obtain a better understanding of the apoptotic effect of selenite per se, but of the role of O(2)(*)(-) in initiation and execution of apoptosis.     

Genetic evidence for NAD(P)H:quinone oxidoreductase 1-catalyzed quinone reduction on passage through the mouse pulmonary circulation

The quinones duroquinone (DQ) and coenzyme Q(1) (CoQ(1)) and quinone reductase inhibitors have been used to identify reductases involved in quinone reduction on passage through the pulmonary circulation. In perfused rat lung, NAD(P)H:quinone oxidoreductase 1 (NQO1) was identified as the predominant DQ reductase and NQO1 and mitochondrial complex I as the CoQ(1) reductases. Since inhibitors have nonspecific effects, the goal was to use Nqo1-null (NQO1(-)/(-)) mice to evaluate DQ as an NQO1 probe in the lung. Lung homogenate cytosol NQO1 activities were 97 ± 11, 54 ± 6, and 5 ± 1 (SE) nmol dichlorophenolindophenol reduced·min(-1)·mg protein(-1) for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs, respectively. Intact lung quinone reduction was evaluated by infusion of DQ (50 μM) or CoQ(1) (60 μM) into the pulmonary arterial inflow of the isolated perfused lung and measurement of pulmonary venous effluent hydroquinone (DQH(2) or CoQ(1)H(2)). DQH(2) efflux rates for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs were 0.65 ± 0.08, 0.45 ± 0.04, and 0.13 ± 0.05 (SE) μmol·min(-1)·g dry lung(-1), respectively. DQ reduction in NQO1(+/+) lungs was inhibited by 90 ± 4% with dicumarol; there was no inhibition in NQO1(-/-) lungs. There was no significant difference in CoQ(1)H(2) efflux rates for NQO1(+/+) and NQO1(-/-) lungs. Differences in DQ reduction were not due to differences in lung dry weights, wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total DQ recoveries. The data provide genetic evidence implicating DQ as a specific NQO1 probe in the perfused rodent lung.     

Metabolism of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate in the neuronal and glial compartments of the adult rat brain as detected by [(13)C, (2)H] NMR spectroscopy

Ex vivo ?(13)C, (2)H? NMR spectroscopy allowed to estimate the relative sizes of neuronal and glial glutamate pools and the relative contributions of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate to the neuronal and glial tricarboxylic acid cycles of the adult rat brain. Rats were infused during 60 min in the right jugular vein with solutions containing (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose or (2-(13)C, 2-(2)H(3)) acetate only. At the end of the infusion the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by high resolution (13)C NMR spectroscopy (90.5 MHz). The relative sizes of the neuronal and glial glutamate pools and the contributions of acetyl-CoA molecules derived from (2-(13)C, (2)H(3)) acetate or (1-(13)C) glucose entering the tricarboxylic acid cycles of both compartments, could be determined by the analysis of (2)H-(13)C multiplets and (2)H induced isotopic shifts observed in the C4 carbon resonances of glutamate and glutamine. During the infusions with (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose, the glial glutamate pool contributed 9% of total cerebral glutamate being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (4%), (2-(13)C) acetyl-CoA (3%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (2%). The neuronal glutamate pool accounted for 91% of the total cerebral glutamate being mainly originated from (2-(13)C) acetyl-CoA (86%) and (2-(13)C, 2-(2)H) acetyl-CoA (5%). During the infusions of (2-(13)C, 2-(2)H(3)) acetate only, the glial glutamate pool contributed 73% of the cerebral glutamate, being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (36%), (2-(13)C, 2-(2)H) acetyl-CoA (27%) and (2-(13)C) acetyl-CoA (10%). The neuronal pool contributed 27% of cerebral glutamate being formed from (2-(13)C) acetyl-CoA (11%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (16%). These results illustrate the potential of ?(13)C, (2)H? NMR spectroscopy as a novel approach to investigate substrate selection and metabolic compartmentation in the adult mammalian brain.     

Effect of inhibitors,redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of ¹⁴C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q_B, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q_A to Q_B) occurs via displacement of UQ from the Q_B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q_B to Q^?_B but is practically unaffected by reduction of Q_A to Q^?_A in the absence of Q_B. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q_B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q_B site.     

Cumulant analysis of charge recombination kinetics in bacterial reaction centers reconstituted into lipid vesicles

The kinetics of charge recombination between the primary photoxidized donor (P(+)) and the secondary reduced quinone acceptor (Q(B)(-)) have been studied in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides incorporated into lecithin vesicles containing large ubiquinone pools over the temperature range 275 K = (50 +/- 15) nm). Following these premises, we describe the kinetics of P(+)Q(B)(-) recombination with a truncated cumulant expansion and relate it to P(Q) and to the free energy changes for Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer (DeltaG(AB)(o)) and for quinone binding (DeltaG(bind)(o)) at Q(B). The model accounts well for the temperature and quinone dependence of the charge recombination kinetics, yielding DeltaG(AB)(o) = -7.67 +/- 0.05 kJ mol(-1) and DeltaG(bind)(o) = -14.6 +/- 0.6 kJ mol(-1) at 298 K.