Modified ligands to FA and FB in photosystem I. Proposed chemical rescue of a [4Fe-4S] cluster with an external thiolate in alanine, glycine, and serine mutants of PsaC

The FB and FA electron acceptors in Photosystem I (PS I) are [4Fe-4S] clusters ligated by cysteines provided by PsaC. In a previous study (Mehari, T., Qiao, F., Scott, M. P., Nellis, D., Zhao, J., Bryant, D., and Golbeck, J. H. (1995) J. Biol. Chem. 270, 28108-28117), we showed that when cysteines 14 and 51 were replaced with serine or alanine, the free proteins contained a S = 1/2, [4Fe-4S] cluster at the unmodified site and a mixed population of S = 1/2, [3Fe-4S] and S = 3/2, [4Fe-4S] clusters at the modified site. We show here that these mutant PsaC proteins can be rebound to P700-FX cores, resulting in fully functional PS I complexes. The low temperature EPR spectra of the C14XPsaC.PS I complexes (where X = S, A, or G) show the photoreduction of a wild-type FA cluster and a modified FB' cluster, the latter with g values of 2.115, 1.899, and 1.852 and linewidths of 110, 70, and 85 MHz. Since neither alanine nor glycine contains a suitable side group, an external thiolate provided by beta-mercaptoethanol has likely been recruited to supply the requisite ligand to the [4Fe-4S] cluster. The EPR spectrum of the C51SPsaC.PS I complex differs from that of the C51APsaC.PS I or C51GPsaC.PS I complexes by the presence of an additional set of resonances, which may be derived from the serine oxygen-ligated cluster. In all other mutant PS I complexes, a wild-type spin-coupled interaction spectrum appears when FA and FB are simultaneously reduced. Single turnover flash studies indicate approximately 50% efficient electron transfer to FA/FB in the C14SPsaC.PS I, C51SPsaC.PS I, C14GPsaC.PS I, and C51GPsaC.PS I mutants and less than 40% in the C14APsaC.PS I and C51APsaC.PS I mutants, compared with approximately 76% in the PS I core reconstructed with wild-type PsaC. These data are consistent with the measurements of the rates of cytochrome c6-NADP+ reductase activity, indicating lower rates in the alanine mutants. It is proposed that the chemical rescue of a [4Fe-4S] cluster with a recruited external thiolate at the modified site allows the mutant PsaC proteins to rebind to PS I and to function in forward electron transfer.     

Mutational analysis of photosystem I polypeptides. Role of PsaD and the lysyl 106 residue in the reductase activity of the photosystem I

The ADC4 mutant of the cyanobacterium Synechocystis sp. PCC 6803 was studied to determine the structural and functional consequences of the absence of PsaD in photosystem I. Isolated ADC4 membranes were shown to be deficient in ferredoxin-mediated NADP(+) reduction, even though charge separation between P700 and FA/FB occurred with high efficiency. Unlike the wild type, FB became preferentially photoreduced when ADC4 membranes were illuminated at 15 K, and the EPR line shapes were relatively broad. Membrane fragments oriented in two dimensions on thin mylar films showed that the g tensor axes of FA- and FB- were identical in the ADC4 and wild type strains, implying that PsaC is oriented similarly on the reaction center. PsaC and the FA/FB iron-sulfur clusters are lost more readily from the ADC4 membranes after treatment with Triton X-100 or chaotropic agents, implying a stabilizing role for PsaD. The specific role of Lys106 of PsaD, which can be crosslinked to Glu93 of ferredoxin (Lelong et al. (1994) J. Biol. Chem. 269, 10034-10039), was probed by site-directed mutagenesis. Chemical cross-linking and protease treatment experiments did not reveal any drastic alterations in the conformation of the mutant PsaD proteins. The EPR spectra of FA and FB in membranes of the Lys106 mutants were similar to those of the wild type. Membranes of all Lys106 mutants showed wild type rates of flavodoxin reduction and flavodoxin-mediated NADP+ reduction, but had 10-54% decrease in the ferredoxin-mediated NADP+ reduction rates. This implies that Lys106 is a dispensable component of the docking site on the reducing side of photosystem I and an ionic interaction between Lys106 of PsaD and Glu93 of ferredoxin is not essential for electron transfer to ferredoxin. These results demonstrate that PsaD serves distinct roles in modulating the EPR spectral characteristics of FA and FB, in stabilizing PsaC on the reaction center, and in facilitating ferredoxin-mediated NADP+ photoreduction on the reducing side of photosystem I.     

Spin-lattice relaxation of the phyllosemiquinone radical of photosystem I

The spin-lattice relaxation time (T1) of the phyllosemiquinone anion radical, A1-, of the photosystem I (PSI) reaction center, were measured between 4.5 and 85 K by electron spin-echo spectroscopy. The selective removal of the iron-sulfur centers, FA, FB, and FX, from PSI allowed the measurement of the intrinsic T1 of the A1- radical. The temperature dependence of the intrinsic (T1)-1 for A1- was found to be approximately T1.3 +/- 0.1. The spin-lattice relaxation of the reduced form of iron-sulfur center FX was also measured at low temperatures, in FA/FB-depleted PSI membranes. It was found that the fast-relaxing FX center enhances the spin-lattice relaxation of the phyllosemiquinone due to dipolar coupling. The effect of the reduced forms of FA/FB on the T1 of the phyllosemiquinone was minor compared to the effect of FX. By analyzing the data with a dipolar model in the light of limitations imposed by other information present in the literature, the distance between the phyllosemiquinone and FX in PSI is estimated to be 14.8 +/- 4 A.     

The eight-amino acid internal loop of PSI-C mediates association of low molecular mass iron-sulfur proteins with the P700-FX core in photosystem I

The PSI-C subunit of photosystem I (PS I) shows similarity to soluble 2[4Fe-4S] ferredoxins. PSI-C contains an eight residue internal loop and a 15 residue C-terminal extension which are absent in the ferredoxins. The eight-residue loop has been shown to interact with PSI-A/PSI-B (Naver, H., Scott, M. P., Golbeck, J. H., Moller, B. L., and Scheller, H. V. (1996) J. Biol. Chem. 271, 8996-9001). Four mutant proteins were constructed. Two were modified barley PSI-C proteins, one lacking the loop and the C terminus (PSI-Ccore) and one where the loop replace the C-terminal extension (PSI-CcoreLc-term). Two were modified Clostridium pasteurianum ferredoxins, one with the loop of barley PSI-C and one with both the loop and the C terminus of PSI-C. Wild-type proteins and the mutants were used to reconstitute barley P700-FX cores lacking PSI-C, -D, and-E. Western blotting showed that PSI-CcoreLc-term binds to PS I, whereas PSI-Ccore does not. Without PSI-D the PSI-CcoreLc-term mutant accepts electrons from FX in contrast to PSI-C mutants without the loop. Flash photolysis of P700-FX cores reconstituted with C. pasteurianum ferredoxin showed that only the ferredoxin mutants with the loop accepted electrons from FX. From this, it is concluded that the loop of PSI-C is necessary and sufficient for the association between PS I and PSI-C, and that the loop is functional as an interaction domain even when positioned at the C terminus of PSI-C or on a low molecular mass, soluble ferredoxin.     

Site-directed mutations of the 4Fe-ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus: role of the cluster-coordinating aspartate in physiological electron transfer reactions

Ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus is a monomeric protein (7.5 kDa) that contains a single [4Fe-4S]1+, 2+ cluster. The protein is unusual in that its cluster is coordinated by three Cys and one Asp residue, rather than by the typical four Cys residues. Site-directed mutagenesis has been used to obtain mutant forms in which the cluster-coordinating Asp was replaced by Cys (D14C) and also by Ser (D14S), together with a third mutant (A1K) which contained N-Met-Lys at the N-terminus instead of N-Ala. Analyses using UV-visible absorption, far-UV circular dichroism, and EPR spectroscopy showed that there were no gross structural differences between the native and the three mutant forms and that they each contained a [4Fe-4S] cluster. The reduction potentials, determined by direct electrochemistry (at 23 degrees C, pH 8.0), of the D14S, D14C, and A1K mutants were -490, -422, and -382 mV, respectively, which compare with values of -375 mV for native [4Fe-4S]-containing ferredoxin and -160 mV for the [3Fe-4S]-containing form. The native, D14C, and A1K proteins functioned as electron acceptors in vitroat 80 degrees C for pyruvate ferredoxin oxidoreductase (POR) and aldehyde ferredoxin oxidoreductase (AOR) from P. furiosus using pyruvate and crotonaldehyde as substrates, respectively. The calculated kcat/Km values were similar for the three proteins when ferredoxin reduction was measured either directly by visible absorption or indirectly by coupling ferredoxin reoxidation to the reduction of metronidazole. In contrast, using the D14S mutant and the 3Fe-form of the native ferredoxin as electron acceptors, the activity with AOR was virtually undetectable, and with POR the calculated kcat/Km values were at least 3-fold lower than those obtained with the native (4Fe-), D14C, and A1K proteins. The ability of this 4Fe-ferredoxin to accept electrons from two oxidoreductases of the same organism is therefore not absolutely dependent upon Asp14, as this residue can be effectively replaced by Cys. However, the efficiency of electron transfer is compromised if Asp14 is replaced by Ser, or if the 4Fe-cluster is converted to the 3Fe-form, but Asp14 does not appear to offer any kinetic advantage over the expected Cys.     

Temperature dependence of forward and reverse electron transfer from A1-, the reduced secondary electron acceptor in photosystem I

Electron-transfer reactions following the formation of P700(+)A1- have been studied in isolated Photosystem I complexes from Synechococcus elongatus between 300 and 5 K by flash absorption spectroscopy. (1) In the range from 300 to 200 K, A1- is reoxidized by electron transfer to the iron-sulfur cluster FX. The rate slows down with decreasing temperature, corresponding to an activation energy of 220 +/- 20 meV in this temperature range. Analyzing the temperature dependence of the rate in terms of nonadiabatic electron-transfer theory, one obtains a reorganization energy of about 1 eV and an edge-to-edge distance between A1 and FX of about 8 A assuming the same distance dependence of the electron-transfer rate as in purple bacterial reaction centers. (2) At temperatures below 150 K, different fractions of PS I complexes attributed to frozen conformational substates can be distinguished. A detailed analysis at 77 K gave the following results: (a) In about 45%, flash-induced electron transfer is limited to the formation and decay of the secondary pair P700(+)A1-. The charge recombination occurs with a t1/2 of about 170 micros. (b) In about 20%, the state P700(+)FX- is formed and recombines with complex kinetics (t1/2 = 5-100 ms). (c) In about 35%, irreversible formation of P700(+)FA- or P700(+)FB- is possible. (3) The transition from efficient forward electron transfer at higher temperatures to heterogeneous photochemistry at low temperatures has been investigated in different glass-forming solvents. The yield of forward electron transfer to the iron-sulfur clusters decreases in a narrow temperature interval. The temperature of the half-maximal effect varies between different solvents and appears to be correlated with their liquid to glass transition. It is proposed that reorganization processes in the surroundings of the reactants which are required for the stabilization of the charge-separated state become arrested near the glass transition. This freezing of protein motions and/or solvent reorganization may affect electron-transfer reactions through changes in the free-energy gap and the reorganization energy. (4) The rate of charge recombination between P700(+) and A1- increases slightly (about 1.5-fold) when the temperature is decreased from 300 to 5 K. This charge recombination characterized by a large driving force is much less influenced by the solvent properties than the forward electron-transfer steps from A1- to FX and FA/B.     

Alcohol and other drugs: an assessment of testing and clinical practices in U.S. trauma centers

The PsaC protein binds two 4Fe-4S centers, FA and FB, in the photosystem I (PSI) protein complex. In the T398 strain of Anabaena variabilis ATCC 29413, the psaC gene encoding this protein has been insertionally inactivated by the introduction of a neomycin resistance gene cartridge in the coding region. Photosystem I complex was purified through native gel electrophoresis of beta-dodecyl maltoside solubilized thylakoid membranes from wild-type and T398 strains of Anabaena 29413. The PSI complex from T398 strain retained functionally active P700, the reaction center chlorophylls. Interestingly, purified PSI complex from T398 cells lacked the PsaD, PsaE, and PsaL polypeptides. Western analysis with polyclonal antibodies raised against these proteins indicated that the two stromal exposed polypeptides, PsaD and PsaE, are absent in isolated thylakoid membranes from T398 cells. The PsaL polypeptide could be detected at a level comparable to that in wild-type thylakoid membranes, although it is absent in the PSI preparation from the mutant. These observations suggest that the PsaC protein is essential for the stable association of PsaD and PsaE, two hydrophilic, extrinsic polypeptides. Moreover, PsaL, a hydrophobic protein is loosely associated with PSI and is lost during the isolation of the PSI complex.     

Coordination of the [2Fe-2S] cluster in wild type and molecular variants of Clostridium pasteurianum ferredoxin, investigated by ESEEM spectroscopy

The [2Fe-2S] ferredoxin from Clostridium pasteurianum contains five cysteine residues in positions 11, 14, 24, 56, and 60. This pattern is unique, and a combination of site-directed mutagenesis and spectroscopy is therefore being implemented to identify the ligands of the [2Fe-2S] cluster. The possible involvement of ligands other than cysteine in some molecular variants of this ferredoxin has been considered, histidines being likely candidates. Therefore, the three histidine residues in positions 6, 7, and 90 of the amino acid sequence have been individually and collectively replaced by alanine or valine. The mutated ferredoxins have been purified and were all found to contain [2Fe-2S] clusters of which the UV-visible absorption spectra were identical to that of the wild-type protein. The H6A/H7A/ H90A triply mutated ferredoxin was further characterized by EPR and by ESEEM spectroscopy and was found to differ only marginally from the wild-type protein. The ESEEM spectra of wild-type ferredoxin displayed weak 14N hyperfine interactions at the three principal g-factors of the [2Fe-2S] center. The estimated 14N coupling constants (Aiso = 0.6 MHz; e2qQ approximately 3.3 MHz) indicate that the ESEEM effect is most likely due to 14N from the polypeptide backbone. 2H2O ESEEM spectra showed that the [2Fe-2S] cluster is accessible for exchange with solvent deuterons. ESEEM spectra of the previously characterized C24A and C14A/C24A variants have been recorded and were also found to be very similar to those of the wild-type protein. There was no evidence for coordination of the [2Fe-2S] cluster by [14N]histidine or other 14N nuclei, in either wild-type or mutant forms of the ferredoxin. By these criteria, the environment of the [2Fe-2S] center is not distinguishable from those in plant-type ferredoxins. Non-cysteinyl coordination most probably occurs only in the C14A/C24A variant, which contains no more than three cysteine residues. The data shown here indicate that the fourth ligand of the [2Fe-2S] cluster is neither a histidine residue nor another nitrogenous ligand. The possibility of oxygenic coordination for this molecular variant is discussed.     

Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin:NADP+ reductase for site-specific ferredoxin mutants

A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.     

Photosystem I is an early target of photoinhibition in barley illuminated at chilling temperatures

Light-induced damage to photosystem I (PSI) was studied during low-light illumination of barley (Hordeum vulgare L.) at chilling temperatures. A 4-h illumination period induced a significant inactivation of PSI electron transport activity. Flash-induced P700 absorption decay measurements revealed progressive damage to (a) the iron-sulfur clusters FA and FB, (b) the iron-sulfur clusters FA, FB, and FX, and (c) the phylloquinone A1 and the chlorophyll AO or P700 of the PSI electron acceptor chain. Light-induced PSI damage was also evidenced by partial degradation of the PSI-A and PSI-B proteins and was correlated with the appearance of smaller proteins. Aggravated photodamage was observed upon illumination of barley leaves infiltrated with KCN, which inhibits Cu,Zn-superoxide dismutase and ascorbate peroxidase. This indicates that the photodamage of PSI in barley observed during low-light illumination at chilling temperatures arises because the defense against active oxygen species by active oxygen-scavenging enzymes is insufficient at these specific conditions. The data obtained demonstrate that photoinhibition of PSI at chilling temperatures is an important phenomenon in a cold-tolerant plant species.     

Photosynthetic electron transport involved in PxcA-dependent proton extrusion in Synechocystis sp. Strain PCC6803: effect of pxcA inactivation on CO2, HCO3-, and NO3- uptake

The product of pxcA (formerly known as cotA) is involved in light-induced Na+-dependent proton extrusion. In the presence of 2, 5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO2 fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO2 fixation is inhibited. At pH 8.0, NO3- uptake activity was very low in the pxcA mutant at low [Na+] (approximately 100 microM). At pH 6.5, the pxcA strain did not take up CO2 or NO3- at low [Na+] and showed very low CO2 uptake activity even at 15 mM Na+. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO2, HCO3-, and NO3- is discussed.     

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.     

Removal of the high-potential [4Fe-4S] center of the beta-subunit from Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of site-directed mutated enzymes

The beta-subunit of the nitrate reductase of Escherichia coli contains four groups of Cys residues (I-IV) which are thought to bind the single [3Fe-4S] center and the three [4Fe-4S] centers. The first or second Cys residue of group I was substituted by site-directed mutagenesis with Ala or Ser. Physiological, biochemical, and EPR studies were performed on the mutated enzymes. With small variations, the properties of these mutant enzymes do not differ from one another. They were found to be as abundant and as stably bound to the membrane as the native enzyme, provided the gamma-subunit was present. Although physiological activity was reduced, it was sufficient to allow growth on nitrate. The study of variations in EPR intensity as a function of the redox potential indicated that these enzymes only contained three iron-sulfur centers instead of the usual four in the native enzyme. Spectral EPR analysis showed that the [4Fe-4S] center of high redox potential (center 1, +80 mV) was missing. The loss of this center did not affect the stable integration of the other three centers. The data presented here are in total contrast to those we have reported for each of the other three centers (centers 2-4), the loss of which was detrimental to the integration of all centers and to the integration of the molybdenum cofactor (Augier et al., in press). Taken together, our results demonstrated that the first and second Cys residues of group I are the ligands of the [4Fe-4S] center (center 1, +80 mV) and that this center participates in electron transfer, but is dispensable. On the basis of these results, it is proposed that the [3Fe-4S] center (center 2, +60 mV) also plays a biological role and that in the native enzyme both high-potential centers, centers 1 and 2, contribute independently and in parallel to the electron transfer to the molybdenum cofactor.     

3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.     

Production of [14C]fumonisin B1 by Fusarium moniliforme MRC 826 in corn cultures

Kinetics of growth and fumonisin production by Fusarium moniliforme MRC 826 in corn "patty" cultures were investigated, and a technique was developed for the production of [14C]fumonisin B1 ([14C]FB1) by using L-[methyl-14C]methionine as the precursor. A significant (P < 0.01) correlation exists between fungal growth and FB1 (r = 0.89) and FB2 (r = 0.87) production in corn patties, beginning after 2 days and reaching the stationary phase after 14 days of incubation. [14C]FB1 was produced by adding L-[methyl-14C]methionine daily to cultures during the logarithmic phase of production. Incorporation of the isotope occurred at C-21 and C-22 of the fumonism molecule and was enhanced in the presence of unlabeled L-methionine. Although the concentration of exogenous unlabeled methionine is critical for incorporation of the 14C label, optimum incorporation was achieved by adding 50 mg of unlabeled L-methionine and 200 mu Ci of L-[methyl-14C]methionine to a corn patty (30 g) over a period of 9 days, yielding [14C]FB1 with a specific activity of 36 mu Ci/mmol.     

Molybdopterin radical in bacterial aldehyde dehydrogenases

The EPR spectra of three different molybdoprotein aldehyde dehydrogenases, one purified from Comamonas testosteroni and two purified from Amycolatopsis methanolica, showed in their oxidized state a novel type of signal. These three enzymes contain two different [2Fe-2S] centers, one flavin and one molybdopterin cytosine dinucleotide, as cofactors all of which are expected to be EPR silent in the oxidized state. The new EPR signal is isotropic with g = 2.004 both at X-band and Q-band frequencies, consists of six partially resolved lines, and shows Curie temperature behavior suggesting that the signal is due to an organic radical with S = 1/2. The EPR spectra of Comamonas testosteroni aldehyde dehydrogenase obtained after cultivation in media containing 15NH4Cl and/or after substitution of H2O for D2O show the presence of both nitrogen and proton hyperfine interactions. Simulations of the spectra of the four possible isotope combinations yield a single set of hyperfine coupling constants. The electron spin shows hyperfine interaction with a single I = 1 (0.9 mT) ascribed to a N nucleus, with a single I = 1/2 (1.5 mT) ascribed to one nonexchangeable H nucleus, and with two, exchangeable, identical I = 1/2 spins (0.6 mT) ascribed to two identical exchangeable protons. Taken together, the observations and simulations rule out amino acid residues or flavin as the origin of the radical. The values of the various hyperfine coupling constants are consistent with the properties expected for a molybdenum(VI)-trihydropterin radical in which the N5 atom is engaged in two hydrogen-bonding interactions with the protein. The majority of the electron (spin) density of the radical is located at and around the N5 atom and at the proton bound to the C6 atom of the pterin ring. The EPR spectrum of the molybdopterin radical broadens above 65 K and is no longer detectable above 168 K, indicating that it is not magnetically isolated. The line broadening is ascribed to cross-relaxation with a nearby, rapidly relaxing, oxidized [2Fe-2S] center involving its magnetic S = 1 excited state in this process. The amount of radical was apparently not changed by addition of aldehydes or oxidants, but it disappeared upon reduction by sodium dithionite. Therefore, whether the molybdenum(VI) trihydropterin radical as detected here is a functional intermediate in catalysis remains to be investigated further.     

Analysis of the reaction coordinate of photosynthetic water oxidation by kinetic measurements of 355 nm absorption changes at different temperatures in photosystem II preparations suspended in either H2O or D2O

Flash-induced absorption changes at 355 nm were measured at different temperatures within the range of 2 degrees C S2) = 14 kJ/mol, EA(S2-->S3) = 35 kJ/mol, and EA(S3-->-->S0 + O2) = 21 kJ/mol for theta > 11 degrees C, 67 kJ/mol for theta < 11 degrees C in PS II core complexes dissolved in H2O; (b) replacement of exchangeable protons by deuterons causes only minor changes ( S2, S2 --> S3, and S3 -->--> S0 + O2, respectively. The corresponding values of PS II membrane fragments are 1.3, 1.3, and 1. 4. Based on these results and corresponding EA data reported in the literature for PS II membrane fragments from spinach [Renger, G., & Hanssum, B. (1992) FEBS Lett. 299, 28-32] and PS II particles from the thermophilic cyanobacterium Synechococcus vulcanus Copeland [Koike, H., Hanssum, B., Inoue, Y., & Renger, G. (1987) Biochim. Biophys. Acta 893, 524-533], the reaction coordinate of the redox sequence in the WOC is inferred to be almost invariant to the evolutionary development from cyanobacteria to higher plants. Furthermore, the rather high activation energy of the S2 --> S3 transition provides evidence for a significant structural change coupled with this reaction. Implications for the mechanism of photosynthetic water oxidation are discussed.     

Electrogenicity at the donor/acceptor sides of cyanobacterial photosystem I

To study electrogenesis the photosystem I particles from Synechococcus elongatus were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference generation has been measured by a direct electrometric method in proteoliposomes absorbed on a phospholipid-impregnated collodion film. The photoelectric response has been found to involve three electrogenic stages associated with (i) iron-sulfur center Fx reduction by the primary electron donor P700, (ii) electron transfer between iron-sulfur centers Fx and FA/FB, and (iii) reduction of photo-oxidized P700+ by reduced cytochrome C553. The relative magnitudes of phases (ii) and (iii) comprised about 20% of phase (i).     

A fluorescent temperature probe based on the association between the excited states of 4-(N,N-dimethylamino)benzonitrile and beta-cyclodextrin

A temperature probe based on the fluorescence properties of the two excited states of 4-(N,N-dimethylamino)benzonitrile (DMABN) in equilibrium with beta-cyclodextrin (CD) in aqueous solution is presented. The fluorescence intensity of the Franck-Condon excited state (FB) as a function of temperature shows a straight line with a correlation better than 0.99 in the 283-308 K temperature interval. On the other hand, the fluorescence intensity of the twisted internal charge-transfer state (FA) remains constant in the same temperature interval because the binding of DMABN in the A* state to CD is isoenthalpic and entropy driven. It is found that the FA/FB ratio is independent of the excitation intensity at a specified temperature, shows a linear relationship with temperature, and allows temperature measurements with a resolution of +/- 2.5 K.     

Epibatidine activates muscle acetylcholine receptors with unique site selectivity

We recently showed that at desensitized muscle nicotinic receptors, epibatidine selects by 300-fold between the two agonist binding sites. To determine whether receptors in the resting, activatible state show similar site selectivity, we studied epibatidine-induced activation of mouse fetal and adult receptors expressed in 293 HEK cells. Kinetic analysis of single-channel currents reveals that (-)-epibatidine binds with 15-fold selectivity to sites of adult receptors and 75-fold selectivity to sites of fetal receptors. For each receptor subtype, site selectivity arises solely from different rates of epibatidine dissociation from the two sites. To determine the structural basis for epibatidine selectivity, we introduced mutations into either the gamma or the delta subunit and measured epibatidine binding and epibatidine-induced single-channel currents. Complexes formed by alpha and mutant gamma(K34S+F172I) subunits bind epibatidine with increased affinity compared to alphagamma complexes, whereas the kinetics of alpha2betadeltagamma(K34S+F172I) receptors reveal no change in affinity of the low-affinity site, but increased affinity of the high-affinity site. Conversely, complexes formed by alpha and mutant delta(S36K+I178F) subunits bind epibatidine with decreased affinity compared to alphadelta complexes, whereas the kinetics of alpha2betagammadelta(S36K+I178F) and alpha2betaepsilondelta(S36K+I178F) receptors show markedly reduced sensitivity to epibatidine. The overall data show that epibatidine activates muscle receptors by binding with high affinity to alphagamma and alphaepsilon sites, but with low affinity to the alphadelta site.