Role of D1-His190 in proton-coupled electron transfer reactions in photosystem II: a chemical complementation study

Recent models for water oxidation in photosystem II propose that His190 of the D1 polypeptide facilitates electron transfer from tyrosine YZ to P680+ by accepting the hydroxyl proton from YZ. To test these models, and to further define the role of D1-His190 in the proton-coupled electron transfer reactions of PSII, the rates of P680+ reduction, YZ oxidation, QA- oxidation, and YZ* reduction were measured in PSII particles isolated from several D1-His190 mutants constructed in the cyanobacterium Synechocystis sp. PCC 6803. These measurements were conducted in the absence and presence of imidazole and other small organic bases. In all mutants examined, the rates of P680+ reduction, YZ oxidation, and YZ* reduction after a single flash were slowed dramatically and the rate of QA- oxidation was accelerated to values consistent with the reduction of P680+ by QA- rather than by YZ. There appeared to be little correlation between these rates and the nature of the residue substituted for D1-His190. However, in nearly all mutants examined, the rates of P680+ reduction, YZ oxidation, and YZ* reduction were accelerated dramatically in the presence of imidazole and other small organic bases (e.g., methyl-substituted imidazoles, histidine, methylamine, ethanolamine, and TRIS). In addition, the rate of QA- oxidation was decelerated substantially. For example, in the presence of 100 mM imidazole, the rate of electron transfer from YZ to P680+ in most D1-His190 mutants increased 26-87-fold. Furthermore, in the presence of 5 mM imidazole, the rate of YZ* reduction in the D1-His190 mutants increased to values comparable to that of Mn-depleted wild-type PSII particles in the absence of imidazole. On the basis of these results, we conclude that D1-His190 is the immediate proton acceptor for YZ and that the hydroxyl proton of YZ remains bound to D1-His190 during the lifetime of YZ*, thereby facilitating the reduction of YZ*.     

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase

A new ruthenium-labeled cytochrome c derivative was designed to measure the actual rate of electron transfer to the Trp-191 radical cation and the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(FeIV = O,R.+)}. The H39C,C102T variant of yeast iso-1-cytochrome c was labeled at the single cysteine residue with a tris (bipyridyl)ruthenium(II) reagent to form Ru-39-Cc. This derivative has the same reactivity with CMPI as native yCc measured by stopped-flow spectroscopy, indicating that the ruthenium group does not interfere with the interaction between the two proteins. Laser excitation of the 1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in electron transfer from RuII* to heme c FeIII with a rate constant of 5 x 10(5) s-1, followed by electron transfer from heme c Fe II to the Trp-191 indolyl radical cation in CMPI(FeIV = O,R*+) with a rate constant of k(eta) = 2 x 10(6) s-1. A subsequent laser flash led to electron transfer from heme c to the oxyferryl heme in CMPII-(FeIV = O,R) with a rate constant of k(etb) = 5000 s-1. The location of the binding domain was determined using a series of surface charge mutants of CcP. The mutations D34N, E290N, and A193F each decreased the values of k(eta) and k(etb) by 2-4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc-CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. A mechanism is proposed for reduction of the oxyferryl heme in which internal electron transfer in CMPII(FeIV = O,R) leads to the regeneration of the radical cation in CMPII-(FeIII,R*+), which is then reduced by yCcII. Thus, both steps in the complete reduction of CMPI involve electron transfer from yCcII to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant k(eta) with theoretical predictions indicate that the electron transfer pathway identified in the crystalline yCc-CcP complex is very efficient. Stopped-flow studies indicate that native yCcII initially reduces the Trp-191 radical cation in CMPI with a second-order rate constant ka, which increases from 1.8 x 10(8) M-1 s-1 at 310 mM ionic strength to > 3 x 10(9) M-1 s-1 at ionic strengths below 100 mM. A second molecule of yCcII then reduces the oxyferryl heme in CMPII with a second-order rate constant kb which increases from 2.7 x 10(7) M-1 s-1 at 310 mM ionic strength to 2.5 x 10(8) M-1 s-1 at 160 mM ionic strength. As the ionic strength is decreased below 100 mM the rate constant for reduction of the oxyferryl heme becomes progressively slower as the reaction is limited by release of the product yCcIII from the yCcIII-CMPII complex. Both ruthenium photoreduction studies and stopped-flow studies demonstrate that the Trp-191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.     

Kinetic evidence that a radical transfer pathway in protein R2 of mouse ribonucleotide reductase is involved in generation of the tyrosyl free radical

Class I ribonucleotide reductases consist of two subunits, R1 and R2. The active site is located in R1; active R2 contains a diferric center and a tyrosyl free radical (Tyr.), both essential for enzymatic activity. The proposed mechanism for the enzymatic reaction includes the transport of a reducing equivalent, i.e. electron or hydrogen radical, across a 35-A distance between Tyr. in R2 and the active site in R1, which are connected by a hydrogen-bonded chain of conserved, catalytically essential amino acid residues. Asp266 and Trp103 in mouse R2 are part of this radical transfer pathway. The diferric/Tyr. site in R2 is reconstituted spontaneously by mixing iron-free apoR2 with Fe(II) and O2. The reconstitution reaction requires the delivery of an external reducing equivalent to form the diferric/Tyr. site. Reconstitution kinetics were investigated in mouse apo-wild type R2 and the three mutants D266A, W103Y, and W103F by rapid freeze-quench electron paramagnetic resonance with >/=4 Fe(II)/R2 at various reaction temperatures. The kinetics of Tyr. formation in D266A and W103Y is on average 20 times slower than in wild type R2. More strikingly, Tyr. formation is completely suppressed in W103F. No change in the reconstitution kinetics was found starting from Fe(II)-preloaded proteins, which shows that the mutations do not affect the rate of iron binding. Our results are consistent with a reaction mechanism using Asp266 and Trp103 for delivery of the external reducing equivalent. Further, the results with W103F suggest that an intact hydrogen-bonded chain is crucial for the reaction, indicating that the external reducing equivalent is a H. Finally, the formation of Tyr. is not the slowest step of the reaction as it is in Escherichia coli R2, consistent with a stronger interaction between Tyr. and the iron center in mouse R2. A new electron paramagnetic resonance visible intermediate named mouse X, strikingly similar to species X found in E. coli R2, was detected only in small amounts under certain conditions. We propose that it may be an intermediate in a side reaction leading to a diferric center without forming the neighboring Tyr.     

Paternal care enhances male reproductive success in pine engraver beetles

His117 of the D2 protein of photosystem II (PS II) is a conserved residue in the second transmembrane region of the protein and has been suggested to bind chlorophyll. Nine site-directed mutations were introduced at residue 117, using both photosystem I (PS I)-containing and PS I-less background strains of the cyanobacterium Synechocystis sp. PCC 6803. Of these nine, four (H117C, H117M, H117N, and H117T) were photoautotrophic in the PS I-containing background. The other mutants (H117F, H117L, H117P, H117R, and H117Y) did not accumulate appreciable amounts of PS II in their thylakoids. The type of residues that can functionally replace His117 support the notion of His117 serving as a chlorophyll ligand. The properties of the H117N and H117T mutants were characterized in more detail. Whereas the properties of the H117N mutant were close to those of wild type, in the H117T mutant the 77-K fluorescence emission spectrum shows a much smaller amplitude at 695 nm than expected on the basis of the amount of PS II that is present. Moreover, in H117T, the amount of light needed to half-saturate O2-evolution rates was twofold higher than in the control strain, and the variable fluorescence yield was quenched. However, O2 evolution rates at saturating light intensity and electron-transport kinetics were normal in the mutant. Also, the radical accessory chlorophyll (Chlz+) formed by donation of an electron to the PS-II reaction center could be generated normally by illumination at low temperature in the H117T mutant. We conclude that the chlorophyll associated with residue 117 of the D2 protein is important for efficient excitation transfer between the proximal antenna and the PS II reaction center. A possible mechanism involving a chlorophyll cation to explain the quenching in the H117T mutant is discussed.     

Identification and analysis of genes involved in anaerobic toluene metabolism by strain T1: putative role of a glycine free radical

The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. The tutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.     

Superoxide produced in the heme pocket of the beta-chain of hemoglobin reacts with the beta-93 cysteine to produce a thiyl radical

The role of the beta-93 cysteine residue in the hemoglobin autoxidation process has been delineated by electron paramagnetic resonance. At low temperatures (8 K) after incubation at 235 K, free radical signals were detected. An analysis of the free radical spectrum produced implies that, besides the superoxide radical expected to be formed during autoxidation, an isotropic free radical is produced with a giso of 2.0133. This g value is consistent with that expected for a sulfur radical. Blocking the beta-93 sulfhydryl group with N-ethylmaleimide was found to eliminate the formation of the isotropic radical, but not the superoxide. This finding confirms the assignment of the isotropic radical as a thiyl radical originating from the oxidation of the cysteine SH group. A kinetic analysis of the time course for the formation of both the superoxide and thiyl radicals is consistent with a reversible electron transfer process between superoxide in the heme pocket of the beta-chains and the cysteine residue. This reaction is expected to produce both a thiyl radical and a peroxide. Direct evidence for peroxide production comes from the detection of a transient Fe(III) heme peroxide complex. The significance of the electron transfer process producing a thiyl radical is discussed. It is shown that the formation of the thiyl radical decreases the rate of autoxidation for the beta-chain and reduces heme degradation attributed to the reaction of superoxide with the heme. The insights gained from these low-temperature studies are believed to be relevant to room-temperature autoxidation.     

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation were investigated. Glycation of bovine serum albumin by methylglyoxal generated the protein-bound free radical, probably the cation radical of the cross-linked Schiff base, as observed in the reaction of methylglyoxal with L-alanine (Yim, H.-S., Kang, S.-O., Hah, Y. C., Chock, P. B., and Yim, M. B. (1995) J. Biol. Chem. 270, 28228-28233) or with Nalpha-acetyl-L-lysine. The glycated bovine serum albumin showed increased electrophoretic mobility suggesting that the basic residues, such as lysine, were modified by methylglyoxal. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen, whereas the protein free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like characteristic, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. The glycated bovine serum albumin cross-linked further to the cytochrome c in the absence of methylglyoxal. The cross-linked cytochrome c maintains its oxidation-reduction properties. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free radicals.     

An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan

The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.     

Characterization of cytochrome c free radical reactions with peptides by mass spectrometry

The reactions of horse heart cytochrome c, hydrogen peroxide, and the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid with a series of polypeptides were investigated using mass spectrometry. The mass spectra obtained from these reactions revealed that after a free radical has been generated on the heme-containing protein horse heart cytochrome c, it can be transferred to other biomolecules. In addition, the number of free radicals transferred to the target molecule could be determined. Recipient peptides/proteins that contained a tyrosine and/or tryptophan amino acid residue were most susceptible to free radical transfer. Using tandem mass spectrometry, the location of the 3,5-dibromo-4-nitrosobenzenesulfonic acid radical adduct on the nonapeptide RWIILGLNK was unequivocally determined to be at the tryptophan residue. We also demonstrated that the presence of an antioxidant in the reaction mixture not only inhibits free radical formation on horse heart cytochrome c, but also interferes with the transfer of the free radical, once it has been formed on cytochrome c.     

Identification of histidine 118 in the D1 polypeptide of photosystem II as the axial ligand to chlorophyll Z

Chlorophyll Z (ChlZ) is a redox-active chlorophyll (Chl) which is photooxidized by low-temperature (<100 K) illumination of photosystem II (PSII) to form a cation radical, ChlZ+. This cofactor has been proposed to be an "accessory" Chl in the PSII reaction center and is expected to be buried in the transmembrane region of the PSII complex, but the location of ChlZ is unknown. A series of single-replacement site-directed mutants of PSII were made in which each of two potentially Chl-ligating histidines, D1-H118 or D2-H117, was substituted with amino acids which varied in their ability to coordinate Chl. Assays of the wild-type and mutant strains showed parallel phenotypes for the D1-118 and D2-117 mutants: noncoordinating or poorly coordinating residues at either position decreased photosynthetic competence and impaired assembly of PSII complexes. Only the mutants substituted with glutamine (D1-H118Q and D2-H117Q) had phenotypes comparable to the wild-type strain. The ChlZ+ cation was characterized by low-temperature electron paramagnetic resonance (EPR), near-infrared (IR) absorbance, and resonance Raman (RR) spectroscopies in wild-type, H118Q, and H117Q PSII core complexes. The quantum yield of ChlZ+ formation is the same (approximately 2.5% per saturating flash at 77 K) for wild-type, H118Q, and H117Q, indicating that its efficiency of photooxidation is unchanged by the mutations. Similarly, the EPR and near-IR absorbance spectra of ChlZ+ are insensitive to the mutations made at D1-118 and D2-117. In contrast, the RR signature of ChlZ+ in H118Q PSII, obtained by selective near-IR excitation into the ChlZ+ cation absorbance band, is significantly altered relative to wild-type PSII while the RR spectrum of ChlZ+ in the H117Q mutant remains identical to wild-type. Shifts in the RR spectrum of ChlZ+ in H118Q reflect a change in the structure of the Chl ring, most likely due to a perturbation of the core size and/or extent of doming caused by a change in the axial ligand to Mg(II). Thus, we conclude that the axial ligand to ChlZ is H118 of the D1 polypeptide. Furthermore, we propose that H117 of the D2 polypeptide is the ligand to a homologous redox-inactive accessory Chl which we term ChlD. The Chl Z and D terminology reflects the 2-fold structural symmetry of PSII which is apparent in the redox-active tyrosines, YZ and YD, and the active/inactive branch homology of the D1/D2 polypeptides with the L/M polypeptides of the bacterial reaction center.     

Identification of the site of the globin-derived radical in leghaemoglobins

Reaction of the Fe3+ form of the oxygen-carrying protein leghaemoglobin (MetLb), derived from the root nodules of lupins, with H2O2 is shown to generate, in addition to an iron (IV)-oxo (ferryl) species, a globin radical. This radical has been detected by EPR spectroscopy and is analogous to the species previously observed with the soybean protein. Analysis of the hyperfine coupling constants and g value of the EPR signal, together with computer simulations and the similarity of the observed spectra of that detected with the soybean form suggest that this species is also a tyrosine-derived phenoxyl radical; this species is believed to arise via an electron-transfer process within the protein with an electron being transferred from the tyrosine residue to an initially-generated Compound-1-type species. Comparison of the protein sequences and structures of the two proteins show that there is only one conserved tyrosine residue (at position 133 in the soybean and 138 in the lupin); this is believed to be the site of the phenoxyl radical. The lupin phenoxyl radical reacts with added water-soluble antioxidants and reducing agents which result in repair of the radical; this may be an important protective mechanism in vivo. Analysis of molecular models of the protein structures is in accord with both the assignment of the radical to this conserved tyrosine residue and the observed radical reactivity.     

Generation of triplet and cation-radical bacteriochlorophyll a in carotenoidless LH1 and LH2 antenna complexes from Rhodobacter sphaeroides

The LH1 antenna complex and a native form of the LH2 complex were isolated from the carotenoidless R26 and R26.1 mutants of Rhodobacter sphaeroides by the use of a new detergent, sucrose monocholate. One-color, pump-and-probe transient Raman spectroscopy of these complexes using 351 nm, approximately 50 ps pulses showed the generation of the triplet state of bacteriochlorophyll a (BChl a), whereas measurements using 355 nm, approximately 12 ns pulses showed the generation of BChl a cation radical. Subpicosecond to nanosecond time-resolved absorption spectroscopy using 388 nm, 200 fs pulses for excitation showed rapid (<1 ps) generation of the triplet state and fast decay (<10 ps) of the singlet state of BChl a. Microsecond absorption spectroscopy confirmed the generation of BChl a cation radical. EPR spectroscopy using 532 nm, approximately 5 ns pulses for excitation established the generation of BChl a cation radical. The EPR line width suggested that the unpaired electron is shared by two BChl a molecules. In LH1, the yield of BChl a cation radical per complex was estimated to be about 80% of that in the reaction center, and in LH2 about 50%. Thus, rapid generation of the triplet state, and its subsequent transformation into the cation-radical state of BChl a have been shown to be intrinsic properties of B870 and B850 BChl a assembly in the carotenoidless LH1 and LH2 antenna complexes. In the case of the carotenoid-containing LH2 complex, the triplet states of BChl a and carotenoid (spheroidene) were generated immediately after excitation, but the triplet-state BChl a was quenched efficiently by the carotenoid so that no BChl a cation radical was generated. Thus, the photoprotective function of the carotenoid in this antenna complex is shown.     

Preserved catalytic activity in an engineered ribonucleotide reductase R2 protein with a nonphysiological radical transfer pathway. The importance of hydrogen bond connections between the participating residues

A hydrogen-bonded catalytic radical transfer pathway in Escherichia coli ribonucleotide reductase (RNR) is evident from the three-dimensional structures of the R1 and R2 proteins, phylogenetic studies, and site-directed mutagenesis experiments. Current knowledge of electron transfer processes is difficult to apply to the very long radical transfer pathway in RNR. To explore the importance of the hydrogen bonds between the participating residues, we converted the protein R2 residue Asp237, one of the conserved residues along the radical transfer route, to an asparagine and a glutamate residue in two separate mutant proteins. In this study, we show that the D237E mutant is catalytically active and has hydrogen bond connections similar to that of the wild type protein. This is the first reported mutant protein that affects the radical transfer pathway while catalytic activity is preserved. The D237N mutant is catalytically inactive, and its tyrosyl radical is unstable, although the mutant can form a diferric-oxo iron center and a R1-R2 complex. The data strongly support our hypothesis that an absolute requirement for radical transfer during catalysis in ribonucleotide reductase is an intact hydrogen-bonded pathway between the radical site in protein R2 and the substrate binding site in R1. Our data thus strongly favor the idea that the electron transfer mechanism in RNR is coupled with proton transfer, i.e. a radical transfer mechanism.     

The nature of the excited state of the reaction center of photosystem II of green plants: a high-resolution fluorescence spectroscopy study

We studied the electronically excited state of the isolated reaction center of photosystem II with high-resolution fluorescence spectroscopy at 5 K and compared the obtained spectral features with those obtained earlier for the primary electron donor. The results show that there is a striking resemblance between the emitting and charge-separating states in the photosystem II reaction center, such as a very similar shape of the phonon wing with characteristic features at 19 and 80 cm-1, almost identical frequencies of a number of vibrational modes, a very similar double-Gaussian shape of the inhomogeneous distribution function, and relatively strong electron-phonon coupling for both states. We suggest that the emission at 5 K originates either from an exciton state delocalized over the inactive branch of the photosystem or from a fraction of the primary electron donor that is long-lived at 5 K. The latter possibility can be explained by a distribution of the free energy difference of the primary charge separation reaction around zero. Both possibilities are in line with the idea that the state that drives primary charge separation in the reaction center of photosystem II is a collective state, with contributions from all chlorophyll molecules in the central part of the complex.     

Comparing the rates and the activation parameters for the forward reaction between the triplet state of zinc cytochrome c and cupriplastocyanin and the back reaction between the zinc cytochrome c cation radical and cuproplastocyanin

This is a comparative study of the photoinduced (so-called forward) electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I), between the triplet state of zinc cytochrome c (3Zncyt) and cupriplastocyanin [pc(II)], and the thermal (so-called back) electron-transfer reaction Zncyt+/pc(I) --> Zncyt/pc(II), between the cation (radical) of zinc cytochrome c (Zncyt+) and cuproplastocyanin [pc(I)], which follows it. Both reactions occur between associated (docked) reactants, and the respective unimolecular rate constants are kF and kB. Our previous studies showed that the forward reaction is gated by a rearrangement of the diprotein complex. Now we examine the back reaction and complare the two. We study the effects of temperature (in the range 273.3-302.9 K) and viscosity (in the range 1.00-17.4 cP) on the rate constants and determine enthalpies (DeltaH), entropies (DeltaS), and free energies (DeltaG) of activation. We compare wild-type spinach plastocyanin, the single mutants Tyr83Leu and Glu59Lys, and the double mutant Glu59Lys/Glu60Gln. The rate constant kB for wild-type spinach plastocyanin and its mutants markedly depends on viscosity, an indication that the back reaction is also gated. The activation parameters DeltaH and DeltaS show that the forward and back reactions have similar mechanisms, involving a rearrangement of the diprotein complex from the initial binding configuration to the reactive configuration. The rearrangements of the complexes 3Zncyt/pc(II) and Zncyt+/pc(I) that gate their respective reactions are similar but not identical. Since the back reaction of all plastocyanin variants is faster than the forward reaction, the difference in free energy between the docking and the reactive configuration is smaller for the back reaction than for the forward reaction. This difference is explained by the change in the electrostatic potential on the plastocyanin surface as Cu(II) is reduced to Cu(I). It is the smaller DeltaH that makes DeltaG smaller for the back reaction than for the forward reaction.     

Loss of inhibition by formate in newly constructed photosystem II D1 mutants, D1-R257E and D1-R257M, of Chlamydomonas reinhardtii

Formate is known to cause significant inhibition in the electron and proton transfers in photosystem II (PSII); this inhibition is uniquely reversed by bicarbonate. It has been suggested that bicarbonate functions by providing ligands to the non-heme iron and by facilitating protonation of the secondary plastoquinone QB. Numerous lines of evidence indicate an intimate relationship of bicarbonate and formate binding of PSII. To investigate the potential amino acid binding environment of bicarbonate/formate in the QB niche, arginine 257 of the PSII D1 polypeptide in the unicellular green alga Chlamydomonas reinhardtii was mutated into a glutamate (D1-R257E) and a methionine (DQ-R257M). The two mutants share the following characteristics. (1) Both have a drastically reduced sensitivity to formate. (2) A larger fraction of QA- persists after flash illumination, which indicates an altered equilibrium constant of the reaction QA-QB<-->QA QB-, in the direction of [QA-], or a larger fraction of non-QB centers. However, there appears to be no significant difference in the rate of electron transfer from QA- to QB. (3) The overall rate of oxygen evolution is significantly reduced, most likely due to changes in the equilibrium constant on the electron acceptor side of PSII or due to a larger fraction in non-QB centers. Additional effects on the donor side cannot yet be excluded. (4) The binding affinity for the herbicide DCMU is unaltered. (5) The mutants grow photosynthetically, but at a decreased (approximately 70% of the wild type) level. (6) The Fo level was elevated (approximately 40-50%) which could be due to a decrease in the excitation energy transfer from the antenna to the PSII reaction center, and/or to an increased level of [QA-] in the dark. (7) A decreased (approximately 10%) ratio of F685 (mainly from CP43) and F695 (mainly from CP47) to F715 (mainly from PSI) emission bands at 77 K suggests a change in the antenna complex. Taken together these results lead to the conclusion that D1-R257 with the positively charged side chain is important for the fully normal functioning of PSII and of growth, and is specially critical for the in vivo binding of formate. Several alternatives are discussed to explain the almost normal functioning of the D1-R257E and D1-R257M mutants.     

Electron transfer between QA and QB in photosystem II is thermodynamically perturbed in phototolerant mutants of Synechocystis sp. PCC 6803

Several random mutations have been generated in the psbA2 gene of Synechocystis sp. PCC 6803 [Narusaka, Y., Murakami, A., Saeki, J., Kobayashi, H., and Satoh, K. (1996) Plant Sci. 115, 261-266]. The phototolerant mutant (I6) carrying all the amino acid substitutions in the lumenal side of D1 protein (S322I, I326F, and F328S) and a site-directed mutant of the same phenotype (NDFS) substituted in the stromal side of the protein (N234D and F260S) were characterized by thermoluminescence measurements. We observed (1) no significant differences in their growth rates at either low or high light irradiance, (2) a downshifted B-band in the NDFS mutant, (3) an upshifted Q-band in the I6 mutant, and (4) a damped period four oscillation of thermoluminescence in the B-band of both mutants. By examining the possible implications of these results on the redox properties of the PS II components in the mutants, we concluded that equilibrium constants for sharing an electron between the primary (QA) and secondary acceptor plastoquinones (QB) are decreased in both mutants.     

Isolation and characterizations of quinone analogue-resistant mutants of bo-type ubiquinol oxidase from Escherichia coli

Cytochrome bo is a member of the heme-copper terminal oxidase superfamily and serves as a four-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. To probe the location and structural properties of the ubiquinol oxidation site, we isolated and characterized five or 10 spontaneous mutants resistant to either 2,6-dimethyl-1,4-benzoquinone, 2,6-dichloro-4-nitrophenol, or 2,6-dichloro-4-dicyanovinylphenol, the potent competitive inhibitors for the oxidation of ubiquinol-1 [Sato-Watanabe, M., Mogi, T., Miyoshi, H., Iwamura, H., Matsushita, K., Adachi, O., and Anraku, Y. (1994) J. Biol. Chem. 269, 28899-28907]. Analyses of the growth yields and the ubiquinol-1 oxidase activities of the mutant membranes showed that the mutations increased the degree of the resistance to the selecting compounds. Notably, several mutants showed the cross-resistance. These data indicate that the binding sites for substrate and the competitive inhibitors are partially overlapped in the ubiquinol oxidation site. All the mutations were linked to the expression vector, and 23 mutations examined were all present in the C-terminal hydrophilic domain (Pro96-His315) of subunit II. Sequencing analysis revealed that seven mutations examined are localized near both ends of the cupredoxin fold. Met248Ile, Ser258Asn, Phe281Ser, and His284Pro are present in a quinol oxidase-specific (Qox) domain and proximal to low-spin heme b in subunit I and the lost CuA site in subunit II, whereas Ile129Thr, Asn198Thr, and Gln233His are rather scattered in a three-dimensional structure and closer to transmembrane helices of subunit II. Our data suggest that the Qox domain and the CuA end of the cupredoxin fold provide the quinol oxidation site and are involved in electron transfer to the metal centers in subunit I.     

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of alpha-melanotropin by NMR

Solution conformation of cyclo(Gly1-His2-Phe3-Arg4-Trp5-Gly6) and its D-Phe analog corresponding to the message sequence [Gly-alpha-MSH5-10] of alpha-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d6 solution and in a DMSO-d6/H2O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the D-analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two gamma-turns, a gamma-turn and a beta-turn, two beta-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a gamma-bend at Gly6, two gamma-bends at Phe3 and Gly6 and a conformer with a single beta-turn and a gamma-bend for the L-Phe analog. On the other hand, a conformation with two fused beta-turns around the two tetrads His2-D-Phe3-Arg4-Trp5 and Trp5-Gly6-Gly1-His2 dominates the equilibrium mixture for the D-Phe analog. For the D-Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.     

Gemcitabine 5'-triphosphate is a stoichiometric mechanism-based inhibitor of Lactobacillus leichmannii ribonucleoside triphosphate reductase: evidence for thiyl radical-mediated nucleotide radical formation

Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii utilizes adenosylcobalamin and catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. One equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate, F2dCTP, rapidly inactivates RTPR. Analysis of the reaction products reveals that inactivation is accompanied by release of two fluoride ions and 0.84 equiv of 5'-deoxyadenosine and attachment of 1 equiv of corrin covalently to an active-site cysteine residue of RTPR. No cytosine release was detected. Proteolysis of corrin-labeled RTPR with endoproteinase Glu-C and peptide mapping at pH 5.8 revealed that C419 was predominantly modified. The kinetics of the inactivation have been examined by stopped-flow (SF) UV-vis spectroscopy and rapid freeze quench (RFQ) electron paramagnetic resonance (EPR) spectroscopy. Monitoring DeltaA525 nm shows that cob(II)alamin is formed with an apparent kobs of 50 s-1, only 2. 5-fold slower than a similar experiment carried out with cytidine 5'-triphosphate (CTP). The same reaction mixture was thus quenched at times from 22 ms to 30 s and examined by EPR spectroscopy. At early time points the EPR spectrum resembled a thiyl radical exchange coupled to cob(II)alamin. From 22 to 255 ms the total spin concentration remained unchanged at 1.4 spins/RTPR, twice that predicted by the amount of cob(II)alamin determined by SF. However, with time the signal attributed to the thiyl radical-cob(II)alamin disappears and new signal(s) with broad feature(s) at g = 2.33 and a sharp feature at g = 2.00 appeared, suggesting formation of cob(II)alamin and a nucleotide-based radical with only dipolar interactions. These studies have been interpreted to support the proposal that an RTPR-based thiyl radical can give rise to a nucleotide-based radical.