Structural and biochemical evidence for an enzymatic quinone redox cycle in Escherichia coli: identification of a novel quinol monooxygenase

Naturally synthesized quinones perform a variety of important cellular functions. Escherichia coli produce both ubiquinone and menaquinone, which are involved in electron transport. However, semiquinone intermediates produced during the one-electron reduction of these compounds, as well as through auto-oxidation of the hydroxyquinone product, generate reactive oxygen species that stress the cell. Here, we present the crystal structure of YgiN, a protein of hitherto unknown function. The three-dimensional fold of YgiN is similar to that of ActVA-Orf6 monooxygenase, which acts on hydroxyquinone substrates. YgiN shares a promoter with "modulator of drug activity B," a protein with activity similar to that of mammalian DT-diaphorase capable of reducing mendione. YgiN was able to reoxidize menadiol, the product of the "modulator of drug activity B" (MdaB) enzymatic reaction. We therefore refer to YgiN as quinol monooxygenase. Modulator of drug activity B is reported to be involved in the protection of cells from reactive oxygen species formed during single electron oxidation and reduction reactions. The enzymatic activities, together with the structural characterization of YgiN, lend evidence to the possible existence of a novel quinone redox cycle in E. coli.     

Absence of large-scale displacement of quinone QB in bacterial photosynthetic reaction centers

Photosynthesis transforms light into chemical energy by coupling electron transfer to proton uptake at the quinone Q(B). The possibility of initiating this process with a brief pulse of light and the known X-ray structure makes the photosynthetic bacterial reaction center a paradigm for studying coupled electron-proton transfer in biology. It has been established that electron transfer from the primary quinone Q(A) to Q(B) is gated by a protein conformational change. On the basis of a dramatic difference in the location of Q(B) in structures derived from crystals cooled to 90 K either under illumination or in the dark, a functional model for the gating mechanism was proposed whereby neutral Q(B) moves 4.5 A before receiving the electron from Q(A)(-) [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science 276, 812-816]. Isotope-edited FTIR difference spectroscopy of Q(B) photoreduction at 290 and 85 K is used to investigate whether Q(B) moves upon reduction. We show that the specific interactions of the carbonyl groups of Q(B) and Q(B)(-) with the protein at a single binding site remain identical at both temperatures. Therefore, the different locations of Q(B) reported in many X-ray crystal structures probably are unrelated to functional electron transfer from Q(A)(-) to Q(B).     

Probing the primary quinone environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy

The photoreduction of the primary electron acceptor, QA, has been characterized by light-induced Fourier transform infrared difference spectroscopy for Rb. sphaeroides reaction centers and for Rsp. rubrum and Rp. viridis chromatophores. The samples were treated both with redox compounds, which rapidly reduce the photooxidized primary electron P+, and with inhibitors of electron transfer from QA- to the secondary quinone QB. This approach yields spectra free from P and P+ contributions which makes possible the study of the microenvironment of QA and QA-.     

Orientation of the primary quinone of bacterial photosynthetic reaction centers contined in chromatophore and reconstituted membranes

The orientation of the reaction center bacteriochlorophyll dimer, (BChl)₂, and primary quinone, Q_I, has been studied by EPR in chromatophores of Rhodopseudomonas sphaeroides R26 and Chromatium vinosum and in the reconstituted membrane multilayers of the isolated Rps. sphaeroides reaction center protein. The similarity in the angular dependence of the (BChl)₂ triplet and Q_I?Fe²⁺ signals in the chromatophore and reconstituted reaction center membrane multilayers indicates that the reaction center is similarly oriented in both native and model membranes. The principle magnetic axes of the (BChl)₂ triplet are found to lie with the x direction approximately parallel to the plane of the membrane surface, and the z and y directions approx. 10–20° away from the plane of the membrane surface and membrane normal, respectively. The Q_I?Fe²⁺ signals are found to have the g 1.82 component positioned perpendicular to the plane of the membrane surface, with an orthogonal low-field transition (at g 1.68 in Rps. Sphaeroides and at g 1.62 in C. vinosum) lying parallel to the plane of the membrane surface. The orientation of Q_I was determined by the angular dependence of this signal in Fe²⁺-depleted reaction center reconstituted membrane multilayers, and it was found to be situated most likely with the plane of the quinone ring perpendicular to the plane of the membrane surface.     

The pathway of the quinol/quinone transhydrogenation reaction in ubiquinol: cytochrome-c reductase of Neurospora mitochondria

Ubiquinol: cytochrome-c reductase, isolated from Neurospora mitochondria as a protein/Triton X-100 preparation, and reconstituted into phospholipid membranes, catalyses the electron transfer from duroquinol to 2.3-dimethoxy-5-decyl-6-methyl-benzoquinone (decQ) on a myxothiazol-insensitive, but antimycin-sensitive, ping-pong pathway. Duroquinol reacts first to form the altered, reduced enzyme E'. This reaction is followed by dissociation of duroquinone making way for E' to bind decQ and convert it into decQH2.     

Proteomic characterization of the Rhodobacter sphaeroides 2.4.1 photosynthetic membrane: identification of new proteins

The Rhodobacter sphaeroides intracytoplasmic membrane (ICM) is an inducible membrane that is dedicated to the major events of bacterial photosynthesis, including harvesting light energy, separating primary charges, and transporting electrons. In this study, multichromatographic methods coupled with Fourier transform ion cyclotron resonance mass spectrometry, combined with subcellular fractionation, was used to test the hypothesis that the photosynthetic membrane of R. sphaeroides 2.4.1 contains a significant number of heretofore unidentified proteins in addition to the integral membrane pigment-protein complexes, including light-harvesting complexes 1 and 2, the photochemical reaction center, and the cytochrome bc(1) complex described previously. Purified ICM vesicles are shown to be enriched in several abundant, newly identified membrane proteins, including a protein of unknown function (AffyChip designation RSP1760) and a possible alkane hydroxylase (RSP1467). When the genes encoding these proteins are mutated, specific photosynthetic phenotypes are noted, illustrating the potential new insights into solar energy utilization to be gained by this proteomic blueprint of the ICM. In addition, proteins necessary for other cellular functions, such as ATP synthesis, respiration, solute transport, protein translocation, and other physiological processes, were also identified to be in association with the ICM. This study is the first to provide a more global view of the protein composition of a photosynthetic membrane from any source. This protein blueprint also provides insights into potential mechanisms for the assembly of the pigment-protein complexes of the photosynthetic apparatus, the formation of the lipid bilayer that houses these integral membrane proteins, and the possible functional interactions of ICM proteins with activities that reside in domains outside this specialized bioenergetic membrane.     

Probing the secondary quinone (QB) environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy

The photoreduction of the secondary electron acceptor, QB, has been characterized by light-induced Fourier transform infrared difference spectroscopy of Rb. sphaeroides and Rp. viridis reaction centers. The reaction centers were supplemented with ubiquinone (UQ10 or UQ0). The QB- state was generated either by continuous illumination at very low intensity or by single flash in the presence of redox compounds which rapidly reduce the photooxidized primary electron donor P+. This approach yields spectra free from P and P+ contributions making possible the study of the microenvironment of QB and QB-. Assignments are proposed for the C...O vibration of QB- and tentatively for the C = O and C = C vibrations of QB.     

Proton NMR study of the heme environment in bacterial quinol oxidases

The heme environment and ligand binding properties of two relatively large membrane proteins containing multiple paramagnetic metal centers, cytochrome bo3 and bd quinol oxidases, have been studied by high field proton nuclear magnetic resonance (NMR) spectroscopy. The oxidized bo3 enzyme displays well-resolved hyperfine-shifted 1H NMR resonance assignable to the low-spin heme b center. The observed spectral changes induced by addition of cyanide to the protein were attributed to the structural perturbations on the low-spin heme (heme b) center by cyanide ligation to the nearby high-spin heme (heme o) of the protein. The oxidized hd oxidase shows extremely broad signals in the spectral region where protons near high-spin heme centers resonate. Addition of cyanide to the oxidized bd enzyme induced no detectable perturbations on the observed hyperfine signals, indicating the insensitive nature of this heme center toward cyanide. The proton signals near the low-spin heme b558 center are only observed in the presence of 20% formamide, consistent with a critical role of viscosity in detecting NMR signals of large membrane proteins. The reduced bd protein also displays hyperfine-shifted 1H NMR signals, indicating that the high-spin heme centers (hemes b595 and d) remain high-spin upon chemical reduction. The results presented here demonstrate that structural changes of one metal center can significantly influence the structural properties of other nearby metal center(s) in large membrane paramagnetic metalloproteins.     

Electron transport dynamics at the quinone acceptor site of bacterial photosynthetic reaction centers as probed using fast temperature changes

Methods of laser-induced temperature jumps and fast freezing were used for testing the rates of thermoinduced conformational transitions of reaction center (RC) complexes in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria. An electron transfer reaction from primary to secondary quinone acceptors was used as a probe of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. To investigate the dynamics of spontaneous decay of the RC state induced by the thermal pulse, the thermal pulse was applied either before or during photoinduced activation of electron transport reactions in the RC acceptor complex. The maximum effect was observed if the thermal pulse was applied against the background of steady-state photoactivation of the RC. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. Independent support of the estimates was obtained from experiments with varied cooling rates of the samples tested.     

Homogeneous catalytic hydrogenation of lipids in the photosynthetic membrane: Effects on membrane structure and photosynthetic activity

We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the homogeneous catalytic hydrogenation of the unsaturated fatty acid residues of membrane lipids. The modified membrane was investigated by electron microscopy, electron-spin resonance and fluorescence polarization methods. Alteration in the functional characteristics of the hydrogenated membrane was monitored by the measurement of photophosphorylation and electron-transport activities. The following results were found. (a) Saturation of 10% of the fatty acyl double bonds induced a definite decrease in the dimension of both thylakoids and loculi. Microdensitometry showed that these structural changes arose from a thickening of the single membranes with a simultaneous decrease in the spacing between membranes. These changes might be accounted for by the alignment of the hydrocarbon chains of saturated lipids and the increased hydrophobicity of the membranes. (b) The orientational pattern of chlorophyll-a molecules was not altered by saturating up to 50% of fatty acyl double bonds in membrane lipids, indicating that the energy-transfer processes amongst the chlorophyll molecules remained functional after hydrogenation. (c) Saturation of double bonds of lipids inhibited whole electron transport prior to the inhibition of Photosystem II and Photosystem I activity, which may suggest that the unsaturation level of fatty acids plays a crucial role by ensuring the lateral mobility of plastoquinone between Photosystem II and Photosystem I.     

Photoreduction of menaquinone in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

Photochemically active reaction centers were isolated from the facultatively aerobic gliding green bacterium Chloroflexus aurantiacus. The absorption difference spectrum, obtained after a flash, reflected the oxidation of P-865, the primary donor, and agreed with that observed in a purified membrane preparation from the same organism (Bruce, B.D., Fuller, R.C. and Blankenship, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6532–6536). By analysis of the kinetics in the presence of reduced N-methylphenazonium methosulfate to prevent accumulation of oxidized P-865, the absorption difference spectrum of an electron acceptor was obtained. The electron acceptor was identified as menaquinone (vitamin K-2), which is reduced to the semiquinone anion in a stoichiometry of approximately one molecule per reaction center. Reduction of menaquinone was accompanied by changes in pigment absorption in the infrared region. Our results indicate that the electron-acceptor chain of C. aurantiacus is very similar to that of purple bacteria.     

Structure of a bacterial photosynthetic membrane: integrity of reaction centers following proteolysis and detergent solubilization

cDNAs were molecularly cloned for proteins specifically expressed in embryo as well as in a chemically induced rat pancreatic B cell tumor in which virally related oncogenes such as v-myc, v-src, v-yes, v-mos and v-kis were previously demonstrated not to be expressed. A plasmid cDNA library consisting of 48,000 independent colonies was constructed from poly(A) containing cytoplasmic RNA isolated from 12 day rat embryo. The library was screened by hybridization with 32p-labelled cDNA synthesized from poly(A) containing RNA of rat pancreatic B cell tumor or normal islet B cells. Two clones were obtained which showed a clearly positive reaction only with tumor probe. Nucleotide sequence of one of them harboring insert of 615 nucleotides was determined and its amino acid sequence of 119 residues was deduced, which showed that the protein encoded by this mRNA is highly basic, basic residues/acidic residues being 1.63.     

Electron acceptors of bacterial photosynthetic reaction centers II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex

The photoreduction of ubiquinone in the electron acceptor complex (Q₁Q₁₁) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H⁺ in the reduction was revealed by the pH dependence of the electron transfer events and by net H⁺ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash Q₁₁ receives an electron via Q₁ to form a stable ubisemiquinone anion (Q?^?₁₁); the second flash generates Q?^?₁. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H⁺, to produce Q₁₁H₂. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H⁺ uptake. Above pH 6 there is a progressive increase in H⁺ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H⁺ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to Q?^?₁₁. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H⁺ binding electron transfer following the second flash.     

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.     

Time-resolved infrared spectroscopy in the study of photosynthetic systems

Time-resolved (TR) infrared (IR) spectroscopy in the nanosecond to second timescale has been extensively used, in the last 30 years, in the study of photosynthetic systems. Interesting results have also been obtained at lower time resolution (minutes or even hours). In this review, we first describe the used techniques—dispersive IR, laser diode IR, rapid-scan Fourier transform (FT)IR, step-scan FTIR—underlying the advantages and disadvantages of each of them. Then, the main TR-IR results obtained so far in the investigation of photosynthetic reactions (in reaction centers, in light-harvesting systems, but also in entire membranes or even in living organisms) are presented. Finally, after the general conclusions, the perspectives in the field of TR-IR applied to photosynthesis are described.     

Fluorescence quenching studies on the characterization of energy generated at the NADH:quinone oxidoreductase and quinol oxidase segments of marine bacteria

Generation of membrane potential (inside-positive) and delta pH (inside-acidic) at two kinds of NADH:quinone oxidoreductase segments, the Na(+)-motive segment and another segment, of Vibrio alginolyticus was examined by monitoring the quenching of fluorescence of oxonol V and that of quinacrine, respectively, with inside-out membrane vesicles. Transient generation of membrane potential at the segment occurred when ubiquinone-1 was added in the presence of KCN and NADH. The membrane potential was resistant to a proton conductor, carbonylcyanide m-chlorophenylhydrazone, indicating that the membrane potential was generated specifically at the Na(+)-motive segment. On the other hand, neither membrane potential nor delta pH was generated at another segment. The Na(+)-motive segment did not generate delta pH, indicating that only Na+ is extruded at this segment. Furthermore, generation of membrane potential and delta pH at the NADH:quinone oxidoreductase segment of V. anguillarum was examined by using the fluorescence quenching technique. This segment of the bacterium was also found to generate delta psi by the extrusion of Na+ but not H+. These results revealed that the fluorescence quenching technique is useful for the rapid identification and characterization of the respiratory segment involved in Na+ translocation.     

Occurrence of menaquinone as the sole isoprenoid quinone in the photosynthetic bacterium Heliobacterium chlorum

Thin-layer chromatography, ultraviolet spectrophotometry, high-performance liquid chromatography, and mass spectroscopy revealed that Heliobacterium chlorum contained menaquinone as the sole quinone with an average amount of 0.35 mol per g dry weight of cells. A menaquinone homologue with nine isoprene units occurred as the major component.Non-standard abbreviations MK-n menaquinone with n isoprene units - TLC thin-layer chromatography - HPLC high-performance liquid chromatography     

Fourier transform infrared evidence of proton uptake by glutamate L212 upon reduction of the secondary quinone QB in the photosynthetic reaction center from Rhodobacter capsulatus

The photoreduction of the secondary quinone Q(B) in native reaction centers (RCs) of Rhodobacter capsulatus and in RCs from the GluL212 --> Gln and GluL212 --> Ala mutants has been investigated at pH 7 in (1)H(2)O and (2)H(2)O by light-induced Fourier transform infrared (FTIR) difference spectroscopy. The Q(B)(-)/Q(B) FTIR difference spectra reflect changes of quinone-protein interactions and of protonation state of carboxylic acid groups as well as reorganization of the protein upon electron transfer. Comparison of Q(B)(-)/Q(B) spectra of native and mutant RCs indicates that the interactions between Q(B) or Q(B)(-) and the protein are similar in all RCs. A differential signal at approximately 1650/1640 cm(-1), which is common to all the spectra, is associated with a movement of a peptide carbonyl or a side chain following Q(B) reduction. On the other hand, Q(B)(-)/Q(B) spectra of native and mutant RCs display several differences, notably between 1700 and 1650 cm(-1) (amide I and side chains), between 1570 and 1530 cm(-1) (amide II), and at 1728-1730 cm(-1) (protonated carboxylic acid groups). In particular, the latter region in native RCs is characterized by a main positive band at 1728 cm(-1) and a negative signal at 1739 cm(-1). In the L212 mutants, the amplitude of the positive band is strongly decreased leading to a differential signal at 1739/1730 cm(-1) that is insensitive to (1)H/(2)H isotopic exchange. In native RCs, only the 1728 cm(-1) band is affected in (2)H(2)O while the 1739 cm(-1) signal is not. The effects of the mutations and of (1)H/(2)H exchange on the Q(B)(-)/Q(B) spectra concur in the attribution of the 1728 cm(-1) band in native RCs to (partial) proton uptake by GluL212 upon the first electron transfer to Q(B), as previously observed in Rhodobacter sphaeroides RCs [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., M?ntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732]. More generally, strong homologies of the Q(B) to Q(B)(-) transition in the RCs from Rb. sphaeroides and Rb. capsulatus are detected by differential FTIR spectroscopy. The FTIR data are discussed in relation with the results from global proton uptake measurements and electrogenic events concomitant with the reduction of Q(B) and with a model of the Q(B) turnover in Rb. sphaeroides RCs [Mulkidjanian, A. Y. (1999) FEBS Lett. 463, 199-204].