BBA - Bioenergetics (v.1787, #10)

Photosysthetic cleavage of water molecules to molecular oxygen is a crucial process for all aerobic life on the Earth. Light-driven oxidation of water occurs in photosystem II (PSII) — a pigment–protein complex embedded in the thylakoid membrane of plants, algae and cyanobacteria. Electron transport across the thylakoid membrane terminated by NADPH and ATP formation is inadvertently coupled with the formation of reactive oxygen species (ROS). Reactive oxygen species are mainly produced by photosystem I; however, under certain circumstances, PSII contributes to the overall formation of ROS in the thylakoid membrane. Under limitation of electron transport reaction between both photosystems, photoreduction of molecular oxygen by the reducing side of PSII generates a superoxide anion radical, its dismutation to hydrogen peroxide and the subsequent formation of a hydroxyl radical terminates the overall process of ROS formation on the PSII electron acceptor side. On the PSII electron donor side, partial or complete inhibition of enzymatic activity of the water-splitting manganese complex is coupled with incomplete oxidation of water to hydrogen peroxide. The review points out the mechanistic aspects in the production of ROS on both the electron acceptor and electron donor side of PSII.
Keywords: Superoxide anion radical; Hydrogen peroxide; Hydroxyl radical; Photosystem II; Redox potential;

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize by Berenika Pokorska; Maksymilian Zienkiewicz; Marta Powikrowska; Anna Drozak; Elzbieta Romanowska (1161-1169).
Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII–LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII–LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.
Keywords: D1 protein turnover; Maize (Zea mays); Thylakoid protein phosphorylation; Thylakoid protease; Photoinhibition;

Spectral characteristic of fluorescence induction in a model cyanobacterium, Synechococcus sp. (PCC 7942) by Radek Kaňa; Ondřej Prášil; Ondřej Komárek; George C. Papageorgiou; Govindjee (1170-1178).
We present here three-dimensional time-wavelength-intensity displays of changes in variable fluorescence, during the O(JI)PSMT transient, observed in cyanobacterium at room temperature. We were able to measure contributions of individual chromophores to fluorescence spectra at various times of fluorescence induction (FI). The method was applied to a freshwater cyanobacterium, Synechococcus sp. (PCC 7942). Analysis of our experimental results provides the following new conclusions: (i) the main chlorophyll (Chl) a emission band at ∼ 685 nm that originates in Photosystem (PS) II exhibits typical fast (OPS) and slow (SMT) FI kinetics with both orange (622 nm) and blue (464 nm) excitation. (ii) Similar kinetics are exhibited for its far-red emission satellite band centered at ∼ 745 nm, where the PS II contribution predominates. (iii) A significant OPS-SMT-type kinetics of C-phycocyanin emission at ∼ 650 nm are observed with the blue light excitation, but not with orange light excitation where the signal rose only slightly to a maximum. The induction of F650 was not caused by an admixture of the F685 fluorescence and thus our data show light-inducible and dark-reversible changes of phycobilin fluorescence in vivo. We discuss possible interpretations of this new observation.
Keywords: Fluorescence induction; Cyanobacterium; PCC 7942; Phycobilisome; State transition;

Identification and characterization of a cytochrome b559 Synechocystis 6803 mutant spontaneously generated from DCMU-inhibited photoheterotrophical growth conditions by Yi-Fang Chiu; Wen-Ching Lin; Chia-Ming Wu; Yung-Han Chen; Chung-Hsien Hung; Shyue-Chu Ke; Hsiu-An Chu (1179-1188).
We identified a spontaneously generated mutant from Synechocystis sp. PCC6803 wild-type cells grown in BG-11 agar plates containing 5 mM Glu and 10 μM DCMU. This mutant carries an R7L mutation on the α-subunit of cyt b559 in photosystem II (PSII). In the recent 2.9 Å PSII crystal structural model, the side chain of this arginine residue is in close contact with the heme propionates of cyt b559. We called this mutant WR7Lα cyt b559. This mutant grew at about the same rate as wild-type cells under photoautotrophical conditions but grew faster than wild-type cells under photoheterotrophical conditions. In addition, 77 K fluorescence and 295 K chlorophyll a fluorescence spectral results indicated that the energy delivery from phycobilisomes to PSII reaction centers was partially inhibited or uncoupled in this mutant. Moreover, WR7Lα cyt b559 mutant cells were more susceptible to photoinhibition than wild-type cells under high light conditions. Furthermore, our EPR results indicated that in a significant fraction of mutant reaction centers, the R7Lα cyt b559 mutation induced the displacement of one of the axial histidine ligands to the heme of cyt b559. On the basis of these results, we propose that the Arg7Leu mutation on the α-subunit of cyt b559 alters the interaction between the APC core complex and PSII reaction centers, which reduces energy delivery from the antenna to the reaction center and thus protects mutant cells from DCMU-induced photo-oxidative stress.
Keywords: Photosystem II; Cytochrome b559; Photoinhibition; Chlorophyll a fluorescence; Site-directed mutagenesis; Synechocystis; EPR;

Ultrafast fluorescence study on the location and mechanism of non-photochemical quenching in diatoms by Yuliya Miloslavina; Irina Grouneva; Petar H. Lambrev; Bernard Lepetit; Reimund Goss; Christian Wilhelm; Alfred R. Holzwarth (1189-1197).
The diatom algae, responsible for at least a quarter of the global photosynthetic carbon assimilation in the oceans, are capable of switching on rapid and efficient photoprotection, which helps them cope with the large fluctuations of light intensity in the moving waters. The enhanced dissipation of excess excitation energy becomes visible as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Intact cells of the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum, which show different NPQ induction kinetics under high light illumination, were investigated by picosecond time-resolved fluorescence under dark and NPQ-inducing high light conditions. The fluorescence kinetics revealed that there are two independent sites responsible for NPQ. The first quenching site is located in an FCP antenna system that is functionally detached from both photosystems, while the second quenching site is located in the PSII-attached antenna. Notwithstanding their different npq induction and reversal kinetics, both diatoms showed identical NPQ via both mechanisms in the steady-state. Their fluorescence decays in the dark-adapted states were different, however. A detailed quenching model is proposed for NPQ in diatoms.
Keywords: Diatom alga; Non-photochemical quenching; Fluorescence kinetic; Ultrafast spectroscopy; Cyclotella; Phaeodactylum;

Functional role of a conserved aspartic acid residue in the motor of the Na+-driven flagellum from Vibrio cholerae by Thomas Vorburger; Andreas Stein; Urs Ziegler; Georg Kaim; Julia Steuber (1198-1204).
The flagellar motor consists of a rotor and a stator and couples the flux of cations (H+ or Na+) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na+-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na+ to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H+-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.
Keywords: Flagellar motor; Motility; PomB; Sodium motive force; Vibrio cholerae;

Mechanism and energetics by which glutamic acid 242 prevents leaks in cytochrome c oxidase by Ville R.I. Kaila; Michael I. Verkhovsky; Gerhard Hummer; Mårten Wikström (1205-1214).
Cytochrome c oxidase (CcO) is the terminal enzyme of aerobic respiration. The energy released from the reduction of molecular oxygen to water is used to pump protons across the mitochondrial or bacterial membrane. The pump function introduces a mechanistic requirement of a valve that prevents protons from flowing backwards during the process. It was recently found that Glu-242, a key amino acid in transferring protons to be pumped across the membrane and to the site of oxygen reduction, fulfils the function of such a valve by preventing simultaneous contact to the pump site and to the proton-conducting D-channel (Kaila V.R.I. et al. Proc. Natl. Acad. Sci. USA 105, 2008). Here we have incorporated the valve model into the framework of the reaction mechanism. The function of the Glu valve is studied by exploring how the redox state of the surrounding metal centers, dielectric effects, and membrane potential, affects the energetics and leaks of this valve. Parallels are drawn between the dynamics of Glu-242 and the long-standing obscure difference between the metastable O H and stable O states of the binuclear center. Our model provides a suggestion for why reduction of the former state is coupled to proton translocation while reduction of the latter is not.
Keywords: Cell respiration; Gating mechanism; Proton leak; Proton translocation; Coupled proton and electron transfer;

Remodeling of tobacco thylakoids by over-expression of maize plastidial transglutaminase by Nikolaos E. Ioannidis; Susana M. Ortigosa; Jon Veramendi; Marta Pintó-Marijuan; Isabel Fleck; Patricia Carvajal; Kiriakos Kotzabasis; Mireya Santos; José M. Torné (1215-1222).
Transglutaminases (TGases, EC are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing ɛ-(γ-glutamyl) links and covalent conjugation of polyamines. In chloroplast it is well established that TGases specifically polyaminylate the light-harvesting antenna of Photosystem (PS) II (LHCII, CP29, CP26, CP24) and therefore a role in photosynthesis has been hypothesised (Della Mea et al. [23] and refs therein). However, the role of TGases in chloroplast is not yet fully understood. Here we report the effect of the over-expression of maize (Zea mays) chloroplast TGase in tobacco (Nicotiana tabacum var. Petit Havana) chloroplasts. The transglutaminase activity in over-expressers was increased 4 times in comparison to the wild-type tobacco plants, which in turn increased the thylakoid associated polyamines about 90%. Functional comparison between Wt tobacco and tgz over-expressers is shown in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both in vivo probing and electron microscopy studies verified thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIα) at the expense of centers located mainly in stroma thylakoids (PSIIβ). A major increase in the granum size (i.e. increase of the number of stacked layers) with a concomitant decrease of stroma thylakoids is reported for the TGase over-expressers.
Keywords: Chloroplast transformation; Grana stacking; Photosystem; Polyamines; Transglutaminase;

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between YZ and P680+. As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F 0.
Keywords: Photosystem II; Oxygen-evolving complex; YZ; CP47; Methylamine; Electron transfer;

Comparison of the electron transport properties of the psbo1 and psbo2 mutants of Arabidopsis thaliana by Yagut Allahverdiyeva; Fikret Mamedov; Maija Holmström; Markus Nurmi; Björn Lundin; Stenbjörn Styring; Cornelia Spetea; Eva-Mari Aro (1230-1237).
Genome sequence of Arabidopsis thaliana (Arabidopsis) revealed two psbO genes (At5g66570 and At3g50820) which encode two distinct PsbO isoforms: PsbO1 and PsbO2, respectively. To get insights into the function of the PsbO1 and PsbO2 isoforms in Arabidopsis we have performed systematic and comprehensive investigations of the whole photosynthetic electron transfer chain in the T-DNA insertion mutant lines, psbo1 and psbo2.The absence of the PsbO1 isoform and presence of only the PsbO2 isoform in the psbo1 mutant results in (i) malfunction of both the donor and acceptor sides of Photosystem (PS) II and (ii) high sensitivity of PSII centers to photodamage, thus implying the importance of the PsbO1 isoform for proper structure and function of PSII. The presence of only the PsbO2 isoform in the PSII centers has consequences not only to the function of PSII but also to the PSI/PSII ratio in thylakoids. These results in modification of the whole electron transfer chain with higher rate of cyclic electron transfer around PSI, faster induction of NPQ and a larger size of the PQ-pool compared to WT, being in line with apparently increased chlororespiration in the psbo1 mutant plants. The presence of only the PsbO1 isoform in the psbo2 mutant did not induce any significant differences in the performance of PSII under standard growth conditions as compared to WT. Nevertheless, under high light illumination, it seems that the presence of also the PsbO2 isoform becomes favourable for efficient repair of the PSII complex.
Keywords: Arabidopsis thaliana; PsbO; Electron transport; Water oxidizing complex; Photosystem;

Dephosphorylation of photosystem II proteins and phosphorylation of CP29 in barley photosynthetic membranes as a response to water stress by Wen-Juan Liu; Yang-Er Chen; Wen-Juan Tian; Jun-Bo Du; Zhong-Wei Zhang; Fei Xu; Fan Zhang; Shu Yuan; Hong-Hui Lin (1238-1245).
Kinetic studies of protein dephosphorylation in barley thylakoid membranes revealed accelerated dephosphorylation of photosystem II (PSII) proteins, and meanwhile rapidly induced phosphorylation of a light-harvesting complex (LHCII) b4, CP29 under water stress. Inhibition of dephosphorylation aggravates stress damages and hampers photosystem recovery after rewatering. This increased dephosphorylation is catalyzed by both intrinsic and extrinsic membrane protein phosphatase. Water stress did not cause any thylakoid destacking, and the lateral migration from granum membranes to stroma-exposed lamellae was only found to CP29, but not other PSII proteins. Activation of plastid proteases and release of TLP40, an inhibitor of the membrane phosphatases, were also enhanced during water stress. Phosphorylation of CP29 may facilitate disassociation of LHCII from PSII complex, disassembly of the LHCII trimer and its subsequent degradation, while general dephosphorylation of PSII proteins may be involved in repair cycle of PSII proteins and stress-response-signaling.
Keywords: Dephosphorylation; Lateral migration; Phosphorylation; Photosystem II; Thylakoid phosphatase;

Heme/heme redox interaction and resolution of individual optical absorption spectra of the hemes in cytochrome bd from Escherichia coli by Dmitry A. Bloch; Vitaliy B. Borisov; Tatsushi Mogi; Michael I. Verkhovsky (1246-1253).
Cytochrome bd is a terminal component of the respiratory chain of Escherichia coli catalyzing reduction of molecular oxygen to water. It contains three hemes, b 558, b 595, and d. The detailed spectroelectrochemical redox titration and numerical modeling of the data reveal significant redox interaction between the low-spin heme b 558 and high-spin heme b 595, whereas the interaction between heme d and either hemes b appears to be rather weak. However, the presence of heme d itself decreases much larger interaction between the two hemes b. Fitting the titration data with a model where redox interaction between the hemes is explicitly included makes it possible to extract individual absorption spectra of all hemes. The α- and β-band reduced-minus-oxidized difference spectra agree with the data published earlier ([22] J.G. Koland, M.J. Miller, R.B. Gennis, Potentiometric analysis of the purified cytochrome d terminal oxidase complex from Escherichia coli, Biochemistry 23 (1984) 1051–1056., and [23] R.M. Lorence, J.G. Koland, R.B. Gennis, Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of “cytochrome a 1” as cytochrome b 595, Biochemistry 25 (1986) 2314–2321.). The Soret band spectra show λ max  = 429.5 nm, λ min  ≈ 413 nm (heme b 558), λ max  = 439 nm, λ min  ≈ 400 ± 1 nm (heme b 595), and λ max  = 430 nm, λ min  = 405 nm (heme d). The spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b; the Soret/α (ΔA 430A 629) ratio for heme d is 1.6.
Keywords: Bacterial metabolism; Chlorin; Molecular bioenergetics; OTTLE; Soret band; Spectral decomposition; Spectroelectrochemistry; α-band; β-band;

Preferential pathways for light-trapping involving β-ligated chlorophylls by Teodor Silviu Balaban; Paula Braun; Christof Hättig; Arnim Hellweg; Jan Kern; Wolfram Saenger; Athina Zouni (1254-1265).
The magnesium atom of chlorophylls (Chls) is always five- or six-coordinated within chlorophyll–protein complexes which are the main light-harvesting systems of plants, algae and most photosynthetic bacteria. Due to the presence of stereocenters and the axial ligation of magnesium the two faces of Chls are diastereotopic. It has been previously recognized that the α-configuration having the magnesium ligand on the opposite face of the 17-propionic acid moiety is more frequently encountered and is more stable than the more seldom β-configuration that has the magnesium ligand on the same face [T.S. Balaban, P. Fromme, A.R. Holzwarth, N. Krauβ, V.I. Prokhorenko, Relevance of the diastereotopic ligation of magnesium atoms in chlorophylls in Photosystem I, Biochim. Biophys. Acta (Bioenergetics), 1556 (2002) 197–207; T. Oba, H. Tamiaki, Which side of the π-macrocycle plane of (bacterio)chlorophylls is favored for binding of the fifth ligand? Photosynth. Res. 74 (2002) 1–10]. In photosystem I only 14 Chls out of a total of 96 are in a β-configuration and these occupy preferential positions around the reaction center. We have now analyzed the α/β dichotomy in the homodimeric photosystem II based on the 2.9 Å resolution crystal structure [A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 Å resolution: role of quinones, lipids, channels and chloride, Nature Struct. Mol. Biol. 16 (2009) 334–342] and find that out of 35 Chls in each monomer only 9 are definitively in the β-configuration, while 4 are uncertain. Ab initio calculations using the approximate coupled-cluster singles-and-doubles model CC2 [O. Christiansen, H. Koch, P. Jørgensen, The second-order approximate coupled cluster singles and doubles model CC2, Chem. Phys. Lett. 243 (1995) 409–418] now correctly predict the absorption spectra of Chls a and b and conclusively show for histidine, which is the most frequent axial ligand of magnesium in chlorophyll–protein complexes, that only slight differences (< 4 nm) are encountered between the α- and β-configurations. Significant red shifts (up to 50 nm) can, however, be encountered in excitonically coupled β–β-Chl dimers. Surprisingly, in both photosystems I and II very similar “special” β–β dimers are encountered at practically the same distances from P700 and P680, respectively. In purple bacteria LH2, the B850 ring is composed exclusively of such tightly coupled β-bacteriochlorophylls a. A statistical analysis of the close contacts with the protein matrix (< 5 Å) shows significant differences between the α- and β-configurations and the subunit providing the axial magnesium ligand. The present study allows us to conclude that the excitation energy transfer in light-harvesting systems, from a peripheral antenna towards the reaction center, may follow preferential pathways due to structural reasons involving β-ligated Chls.
Keywords: Crystal structure; Photosystem I; Photosystem II; Diastereotopicity; Chlorophyll complex; ab initio calculation;

Electrostatics of the FeS clusters in respiratory complex I by Vernon A. Couch; Emile S. Medvedev; Alexei A. Stuchebrukhov (1266-1271).
Respiratory complex I couples the transfer of electrons from NADH to ubiquinone and the translocation of protons across the mitochondrial membrane. A detailed understanding of the midpoint reduction potentials (E m ) of each redox center and the factors which influence those potentials are critical in the elucidation of the mechanism of electron transfer in this enzyme. We present accurate electrostatic interaction energies for the iron–sulfur (FeS) clusters of complex I to facilitate the development of models and the interpretation of experiments in connection to electron transfer (ET) in this enzyme. To calculate redox titration curves for the FeS clusters it is necessary to include interactions between clusters, which in turn can be used to refine E m values and validate spectroscopic assignments of each cluster. Calculated titration curves for clusters N4, N5, and N6a are discussed. Furthermore, we present some initial findings on the electrostatics of the redox centers of complex I under the influence of externally applied membrane potentials. A means of determining the location of the FeS cofactors within the holo-complex based on electrostatic arguments is proposed. A simple electrostatic model of the protein/membrane system is examined to illustrate the viability of our hypothesis.
Keywords: Complex I; NADH dehydrogenase; Iron–sulfur cluster; Electron transfer; Redox potential; Poisson–Boltzmann equation;

Critical structural role of R481 in cytochrome c oxidase from Rhodobacter sphaeroides by Tsuyoshi Egawa; Hyun Ju Lee; Robert B. Gennis; Syun-Ru Yeh; Denis L. Rousseau (1272-1275).
The R481 residue in cytochrome c oxidase from Rhodobacter sphaeroides forms hydrogen bonds with the propionate groups of both heme a and heme a 3. It has been postulated that R481 is the proton loading site in the proton exit pathway essential for proton translocation. A recent functional study showed that the mutations of R481 to His, Leu and Gln cause the reduction of the activity to ∼ 5–18% of the native level, and the absence of proton pumping in R481Q but retention of ∼ 40% efficiency in R481H and R481L (H.J. Lee, L. Öjemyr, A. Vakkasoglu, P. Brzezinski and R. B. Gennis, manuscript submitted). To decipher the molecular mechanism underlying the perturbed functionalities, we have used resonance Raman spectroscopy to examine the structural properties of the three mutants. The data show that the frequencies of the formyl C=    O stretching modes of both the heme a and a 3 in the mutants are characteristic of formyl groups exposed to an aqueous environment, indicating that the mutations disrupt the native H-bonding interaction between the formyl group of heme a and R52, as well as the hydrophobic environment surrounding the formyl group of heme a 3. In addition to the change in the environments of heme a and a 3, the Raman data show that the mutations induce a partial conversion of the heme a 3 from a high-spin to a low-spin state, suggesting that the mutations are associated with the rearrangement of the CuB-heme a 3 binuclear center. The Raman results reported here demonstrate that R481 plays a critical role in supporting efficient proton pumping, by holding the heme groups in a proper environment.
Keywords: Raman scattering; Bioenergetics; Proton translocation; Mutants; Heme;

Inhibition of proton-transfer steps in transhydrogenase by transition metal ions by Simon J. Whitehead; Masayo Iwaki; Nick P.J. Cotton; Peter R. Rich; J. Baz Jackson (1276-1288).
Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a “cyclic reaction” was stimulated. Of metal ions tested they were effective in the order Pb2+  > Cu2+  > Zn2+  = Cd2+  > Ni2+  > Co2+. The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn2+-binding site(s). A mutant in which βHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn2+. Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn2+-induced FTIR difference spectra of the βHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that βHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase.
Keywords: Transhydrogenase; Membrane protein; Zinc ion; Proton translocation; Redox; FTIR;