Nitrogenase IV. Simple method of purification to homogeneity of nitrogenase components from Azotobacter vinelandii

Extracts of Azotobacter vinelandii have been fractionated by simple techniques to obtain highly purified components of nitrogenase. The yield of each component is greater than 60%. Purified Component I has a specific activity of 1638 nmoles ethylene formed/min per mg protein. The spectrum of Component I exhibits a broad absorption between 300 and 600 nm, with no distinctive peaks or shoulders. Addition of sodium dithionite or exposure to air has no effect on the absorption spectrum. Component I, examined at 4.2 °K has EPR signals at g = 4.2, 3.65 and 2.01. Addition of sodium dithionite does not produce additional resonances nor does it alter the signals already present. Crystals of Component I are dark brown and needle-shaped.Purified Component II has a specific activity of 1815 nmoles ethylene formed/min per mg protein. The absorption spectrum has no peaks or shoulders between 390 and 650 nm. Upon exposure of Component II to air, absorption increases between 400 and 650 nm. Treatment of oxidized Component II with dithionite causes this absorption to fall below that of the native Component II. EPR spectra of Component II has signals at g values of 2.05, 1.94, and 1.88. Upon inactivation by O₂, these signals disappear.Neither component by itself has detectable acetylene-reducing or N₂-fixing activity. The ratio of acetylene reduced to N₂ fixed is 3.86 with different ratios of the components. Both components form aggregated species upon exposure to air. Dithionite does not reverse this effect.     

EPR spectra of Photosystem I and other iron protein components in intact cells of cyanobacteria

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe³⁺ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor ‘X’ of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 ± 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe³⁺, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.     

The role of an iron-sulfur center and siroheme in spinach nitrite reductase.

Purified spinach nitrite reductase, a protein that contains siroheme, is characterized by absorption maxima in the visible region at 385 and 573 nm. On addition of the substrate nitrite, the bands shift to 360 and 570 nm. Dithionite also causes shifts in the maxima of the visible absorption region. Electron paramagnetic resonance studies show that the untreated enzyme contains a high-spin Fe³⁺ heme and that the addition of cyanide, an inhibitor that is competitive with nitrite, results in a spin-state change of the heme. Electron paramagnetic resonance analysis of the enzyme in the presence of dithionite or dithionite plus cyanide indicates the presence of a reduced iron-sulfur center with rhombic symmetry (g-values of 2.03, 1.94, and 1.91). In contrast, when the enzyme is treated with dithionite plus nitrite, the EPR spectrum of an NO-heme complex (g-values of 2.07 and 2.00) is observed. The presence of an iron-sulfur center has also been confirmed by chemical analyses of the nonheme iron and acid-labile sulfide in nitrite reductase. These results are discussed in terms of a mechanism for nitrite reduction that involves electron transfer between the iron-sulfur center and siroheme.     

Distribution of Xanthomonas oryzae pv. oryzae DNA Modification Systems in Asia

The presence or absence of two DNA modification systems, XorI and XorII, in 195 strains of Xanthomonas oryzae pv. oryzae collected from different major rice-growing countries of Asia was assessed. All four possible phenotypes (XorI⁺ XorII⁺, XorI⁺ XorII⁻, XorI⁻ XorII⁺ and XorI⁻ XorII⁻) were detected in the population at a ratio of approximately 1:2:2:2. The XorI⁺ XorII⁺ and XorI⁻ XorII⁺ phenotypes were observed predominantly in strains from southeast Asia (Philippines, Malaysia, and Indonesia), whereas strains with the phenotypes XorI⁻ XorII⁻ and XorI⁺ XorII⁻ were distributed in south Asia (India and Nepal) and northeast Asia (China, Korea, and Japan), respectively. Based on the prevalence and geographic distribution of the XorI and XorII systems, we suggest that the XorI modification system originated in northeast Asia and was later introduced to southeast Asia, while the XorII system originated in southeast Asia and moved to northeast Asia and south Asia. Genomic DNA from all tested strains of X. oryzae pv. oryzae that were resistant to digestion by endonuclease XorII or its isoschizomer PvuI also hybridized with a 7.0-kb clone that contained the XorII modification system, whereas strains that were digested by XorII or PvuI lacked DNA that hybridized with the clone. Size polymorphisms were observed in fragments that hybridized with the 7.0-kb clone. However, a single hybridization pattern generally was found in XorII⁺ strains within a country, indicating clonal maintenance of the XorII methyltransferase gene locus. The locus was monomorphic for X. oryzae pv. oryzae strains from the Philippines and all strains from Indonesia and Korea.     

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD⁺ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD⁺ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.     

The orientation of the magnetic axes of membrane-bound iron-sulfur clusters and a cytochrome b-559 in the green halophilic alga Dunaliella parva

Photosynthetic membrane fragments separated from whole cells of the green alga Dunaliella parva, were oriented by incorporation into multilayers on thin Mylar films. These partially dehydrated films were then examined by EPR spectroscopy for evidence of orientation of paramagnetic components. Five previously identified paramagnetic components, the reduced states of iron-sulfur clusters A and B, the intermediate acceptor X^?, the reduced Rieske iron-sulfur cluster, and oxidized cytochrome b-559, displayed EPR signals showing orientation. In addition, several previously unknown paramagnetic components were also observed to be oriented. Four components, previously characterized in spinach chloroplast preparations, the iron-sulfur clusters A and B, the intermediate acceptor X^?, and cytochrome b-559, were shown to be similar in the green alga, D. parva. The orientations of iron-sulfur clusters A and B, however, were determined unambiguously in this preparation; this was not possible in previous work with spinach. The heme plane orientation of cytochrome b-559 was found to be perpendicular to the membrane plane in agreement with the results in spinach preparations. A new photoinduced EPR signal with g values of 1.88, 1.97 and 2.12 was seen only in the oriented preparations and was indicative of a reduced iron-sulfur cluster with an orientation different from that of iron-sulfur cluster A or B. This suggests the existence of a previously unidentified acceptor in Photosystem I of green plants. These studies clearly show that the orientation of these components in bioenergetic membranes are conserved over a large span of evolutionary development and are, therefore, an important aspect of the mechanism of electron transfer.     

Chemical analysis of the female sex pheromone in Palpita nigropunctalis (Lepidoptera: Crambidae)

The lilac pyralid, Palpita nigropunctalis Bremer (Lepidoptera: Crambidae), is a common pest of Oleaceae plants. A crude extract of the female sex pheromone glands was examined by gas chromatography-electroantennogram detection (GC-EAD) and GC coupled to a mass spectrometer (GC/MS). The GC-EAD analysis revealed three EAG-active components (I–III) in a ratio of 1:0.2:0.01 (I: II: III). GC/MS analysis successfully recorded the mass spectra of I and II. For I, ions at m/z 238 (M⁺) and 220 ([M-18]⁺) indicated the structure of a monoenyl aldehyde with a 16-carbon chain. For II, M⁺ was not detected, but ions at m/z 222 ([M-60]⁺) and 61 ([AcOH+1]⁺) suggested that II was a monoenyl acetate with a 16-carbon chain. Further GC/MS analysis of the extract treated with dimethyl disulfide revealed that the double bonds in both I and II are located at the same position of 11th-carbon. In addition, the pheromone extract was examined by GC/Fourier transform-infrared spectrophotometer (GC/FT-IR). An IR spectrum of I showed characteristic absorption at 1716 and 966?cm^?1, indicating a formyl group and E configuration of the double bond, respectively. In the case of II, absorption at 1745 and 968?cm^?1 indicated an ester carbonyl and E configuration, respectively. Taken together and by comparison with authentic standards, I and II were confirmed as (E)-11-hexadecenal and (E)-11-hexadecenyl acetate, respectively; while III was speculated as (E)-11-hexadecen-1-ol. The synthetic I, II and III all coincided well with those of the natural components in chemical data, and elicited strong electroantennographic activity in male P. nigropunctalis.     

Nucleotide sequence and genetic analysis of the Rhodobacter capsulatus ORF6-nifU I SVW gene region: possible role of Nif W in homocitrate processing

DNA sequence analysis of a 3494-bp HindIII-Bc1I fragment of the Rhodobacter capsulatus nif region A revealed genes that are homologous to ORF6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiella pneumoniae. R. capsulatus nifU, which is present in two copies, encodes a novel type of NifU protein. The deduced amino acid sequences of NifU_I and NifU_II share homology only with the C-terminal domain of NifU from A. vinelandii and K. pneurnoniae. In contrast to nifA andnifB which are almost perfectly duplicated, the predicted amino acid sequences of the two NifU proteins showed only 39% sequence identity. Expression of the ORF6-nifU _ISVW operon, which is preceded by a putative σ⁵⁴-dependent promoter, required the function of NifA and the nif-specific rpoN gene product encoded by nifR4. Analysis of defined insertion and deletion mutants demonstrated that only nifS was absolutely essential for nitrogen fixation in R. capsulatus. Strains carrying mutations in nifV were capable of very slow diazotrophic growth, whereas ORF6, nifU _I and nifW mutants as well as a nifU _I/nifU_II, double mutant exhibited a Nif⁺ phenotype. Interestingly, R. capsulatus nifV mutants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative nitrogenase system. Homocitrate added to the growth medium repressed ethane formation and cured the NifV phenotype in R. capsulatus. Higher concentrations of homocitrate were necessary to complement the NifV phenotype of a polar nifV mutant (NifV^?NifW^?), indicating a possible role of NifW either in homocitrate transport or in the incorporation of this compound into the iron-molybdenum cofactor of nitrogenase.     

Polynuclear water-soluble dinitrosyl iron complexes with cysteine or glutathione ligands: Electron paramagnetic resonance and optical studies

Electron paramagnetic resonance and optical spectrophotometric studies have demonstrated that low-molecular dinitrosyl iron complexes (DNICs) with cysteine or glutathione exist in aqueous solutions in the form of paramagnetic mononuclear (М-DNICs) and diamagnetic binuclear complexes (B-DNICs). The latter represent Roussin’s red salt esters and can be prepared by treatment of aqueous solutions of Fe²⁺ and thiols (рН 7.4) with gaseous nitric oxide (NO) at the thiol:Fe²⁺ ratio 1:1. М-DNICs are synthesized under identical conditions at the thiol:Fe²⁺ ratios above 20 and produce an EPR signal with an electronic configuration {Fe(NO)₂}⁷ at g_aver. = 2.03. At neutral pH, aqueous solutions contain both M-DNICs and B-DNICs (the content of the latter makes up to 50% of the total DNIC pool). The concentration of B-DNICs decreases with a rise in pH; at рН 9–10, the solutions contain predominantly M-DNICs. The addition of thiol excess to aqueous solutions of B-DNICs synthesized at the thiol:Fe²⁺ ratio 1:2 results in their conversion into М-DNICs, the total amount of iron incorporated into M-DNICs not exceeding 50% of the total iron pool in B-DNICs. Air bubbling of cys-М-DNIC solutions results in cysteine oxidation-controlled conversion of М-DNICs first into cys-B-DNICs and then into the EPR-silent compound Х able to generate a strong absorption band at 278 nm. In the presence of glutathione or cysteine excess, compound Х is converted into B-DNIC/M-DNIC and is completely decomposed under effect of the Fe²⁺ chelator о-phenanthroline or N-methyl-d-glucamine dithiocarbamate (MGD). Moreover, MGD initiates the synthesis of paramagnetic mononitrosyl iron complexes with MGD. It is hypothesized that compound Х represents a polynuclear DNIC with cysteine, most probably, an appropriate Roussin’s black salt thioesters and cannot be prepared by simple substitution of М-DNIC cysteine for glutathione. Treatment of М-DNIC with sodium dithionite attenuates the EPR signal at g_aver. = 2.03 and stimulates the appearance of an EPR signal at g_aver. = 2.0 with a hypothetical electronic configuration {Fe(NO)₂}⁹. These changes can be reversed by storage of DNIC solutions in atmospheric air. The EPR signal at g_aver. = 2.0 generated upon treatment of B-DNICs with dithionite also disappears after incubation of B-DNIC solutions in air. In all probability, the center responsible for this EPR signal represents М-DNIC formed in a small amount during dithionite-induced decomposition of B-DNIC.     

Role of tfdCIDIEIFI and tfdDIICIIEIIFII Gene Modules in Catabolism of 3-Chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC_ID_IE_IF_I (module I) and tfdD_IIC_IIE_IIF_II (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC_ID_IE_IF_I grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD_IIC_IIE_IIF_II. In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.     

Electron paramagnetic resonance crystallography of spin-labeled hemoglobin-protein fine structures.

Various hemoglobin derivatives have been labeled at the Cys-β93 residue with a bulky and “strongly immobilized” nitroxide maleimide (I) and a smaller, more flexible and “weakly immobilized” nitroxide iodoacetamide (II) and crystallized. The angular dependence of the paramagnetic resonance of the spin-label was measured for the ab, $\text{ac}{}^1$ and $\text{bc}{}^1$ planes at 298 K and 77 K for spin-labeled crystals of Oxyhemoglobin, methemoglobin fluoride, and methemoglobin azide. In the case of the methemoglobin crystals, the angular variation of the heme resonance was also monitored at 77 K. From the hyperfine splitting data, the spin-label I was found to assume specific orientations at both temperatures with some motional narrowing at 298 K. Spin-label II is specifically oriented only at room temperature but is frozen at 77 K in random orientations. Oxyhemoglobin labeled with I (I-HbO₂²) has the most prominent spin-label orientation (z∥b, x∥a) and the less abundant spin-labels with (z∥b ± 15 °) (Ohnishi et al., 1966). The corresponding spin-label orientations for I-Hb⁺ F^? are ($\text{z∥a, x∥c}{}^1$) and ($\text{z∥c}{}^1\text{, x∥a}$). Crystals of I-Hb⁺ N^?₃ have spin-labels oriented along angular directions similar, but not identical to those of I-Hb⁺F^?. Therefore, there are probably significant peptide segmental displacements when HbO₂ is oxidized to methemoglobins. At 25 °C II-Hb⁺ N^?₃ has spin-label orientations not too different from those in I-Hb⁺ N^?₃, whereas in HbO₂ the two spin-labels show significant differences in their orientations.     

The Escherichia coli adenylate cyclase complex. Regulation by enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

The activity of adenylate cyclase of Escherichia coli measured in toluene-treated cells under standard conditions is subject to control by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Sugars such as glucose, which are transported by the PTS, will inhibit adenylate cyclase provided the PTS is functional. An analysis was made of the properties of E. coli strains carrying mutations in PTS proteins. Leaky mutants in the PTS protein HPr are similar to wild-type strains with respect to cAMp regulation; adenylate cyclase activity in toluene-treated cells and intracellular cAMP levels are in the normal range. Furthermore, adenylate cyclase in toluene-treated cells of leaky HPr mutants is inhibited by glucose. In contrast, mutations in the PTS protein Enzyme I result in abnormalities in cAMP regulation. Enzyme I mutants generally have low intracellular cAMP levels. Leaky Enzyme I mutants show an unusual phosphoenolpyruvate-dependent activation of adenylate cyclase that is not seen in Enzyme I⁺ revertants or in Enzyme I deletions. A leaky Enzyme I mutant exhibits changes in the temperature-activity profile for adenylate cyclase, indicating that adenylate cyclase activity is controlled by Enzyme I. Temperature-shift studies suggest a functional complex between adenylate cyclase and a regulator protein at 30 °C that can be reversibly dissociated at 40 °C. These studies further support the model for adenylate cyclase activation that involves phosphoenolpyruvate-dependent phosphorylation of a PTS protein complexed to adenylate cyclase.     

Mitochondrial control of sugar utilization in Saccharomyces cerevisiae.

When a number of wild-type strains of Saccharomyces cerevisiae—all capable of utilizing the three sugars galactose, maltose, and α-methyl-d-glucoside for growth—were converted by ethidium bromide (EtdBr) mutagenesis to stable cytoplasmic petite (rho^?) mutants, the latter lost the ability to grow on one or more of these sugars. The actual pattern of retention (or loss) or sugar utilization by these mutants depended on the wild-type strain, but was independent of the length of exposure to EtdBr during mutagenesis. This treatment varied from 0.5 to 24 h, by which time the majority of the mutants must have been of the mitochondrial (mt) DNA-deficient rho⁰ type. Furthermore, with one exception—involving the ability of one set of mutants to utilize α-methyl-glucoside—all rho^? mutants derived from the same wild type exhibited the same, discrete pattern of sugar utilization. Respiration-deficient mutants with defined lesions in their mtDNA (mit^? mutants) exhibited the same pattern of sugar utilization as did the petite mutants of the same strain. Diploid petite strains also exhibited discrete, but less stringent, patterns of sugar utilization. For any one genotype this pattern was identical whether the mutant was generated by crossing two haploid rho^? strains, themselves derived by EtdBr mutagenesis, or by EtdBr mutagenesis of the diploid obtained from a haploid wild-type × wild-type cross. In such mutant diploids the sugar-positive phenotype was usually dominant, but there were indications in some instances of modulation of this effect by virtue of nuclear gene interactions. Various respiration-deficient mutants incapable of utilizing α-methylglucoside also were unable to form α-glucosidase, but were able to do so after being rendered permeable by exposure to dimethyl sulfoxide. Arguments are advanced that respiring mitochondria generate an entity—probably not directly related to ATP production—required for the expression of nuclear genes or their products, some of which may be necessary for plasma membrane function.     

Diffusion of tetracycline across the outer membrane of Escherichia coli K-12: involvement of protein Ia.

The possibility that passage of tetracycline across the outer membrane of $\text{E.}\text{coli}$ K-12 is controlled by one or more of the proteins I_a, I_b and II¹ (Henning's nomenclature) was investigated. A mutant lacking protein I_a (obtained by selection for resistance to phage TuI_a) was more resistant to tetracycline than wild-type strains or those lacking only proteins I_b or II¹. The envelope protein composition of a tetracycline-resistant mutant ($\text{cmlB}$) was altered in several respects, but the major change involved loss of protein I_a. These data support our previous suggestion [12] that tetracycline diffuses across the outer membrane through hydrophilic regions. Furthermore, they imply that only protein I_a plays a significant role in the passage of this antibiotic across the outer membrane.     

Mutants of Escherichia coli with altered deoxyribonucleases. II. Isolation and characterization of mutants for exonuclease I

Mutants of Escherichia coli K12 deficient in exonuclease I (xon^?)³ were identified by enzymic assay of randomly selected, heavily mutagenized clones. From one of the six mutants of independent origin a thermolabile variant of exonuclease I was partially purified and identified, indicating that the mutation is probably in a structural gene for the enzyme. Transduction of this mutation into a recB^? recC^? strain did not result in the suppression of any of the phenotypic traits of the recipient. Although the five other mutants also appear to have temperature-sensitive exonuclease I activities in crude extracts, these enzymes were not sufficiently stable to permit purification. These latter mutations were of the xonA^? type; they produced a temperature-dependent suppression of the sensitivity to ultraviolet light and to mitomycin C manifested by a recB^? recC^? strain. None of the six mutations were of the sbcB^? type; that is, they did not suppress the recombination deficiency of a recB^? recC^? strain.In experiments with bacteriophage Plke, the six mutations were 41 to 62% cotransducible with the his region of E. coli. Heterozygous F′-merodiploids were constructed and studied for possible complementation of exonuclease I activity. All six mutations and an sbcB^? mutation were recessive to the wild-type alleles, and all were found to belong to a single complementation group. The results suggest that alterations of a structural gene for exonuclease I may result in the indirect suppression of the ultraviolet and mitomycin sensitivity manifested by recB^? recC^? strains.     

Evidence for an anisotropic magnetic interaction between the (bacteriopheophytin) intermediary acceptor and the first quinone acceptor in bacterial photosynthesis

We have investigated electron spin polarization effects occurring in protonated and perdeuterated reaction centers of Rhodospirillum rubrum with electron spin resonance at 9 and 35 GHz (X- and Q-band). As for Rhodopseudomonas sphaeroides strains 2.4.1 and R-26 (Gast, P. and Hoff, A.J. (1979) Biochim. Biophys. Acta 548, 520–535; Gast, P., Mushlin, R.A. and Hoff, A.J. (1982) J. Phys. Chem. 86, 2886–2891), electron spin polarization effects of the prereduced first quinone acceptor Q^?_A in R. rubrum are strongly nonuniform. This nonuniformity is due to an anisotropic magnetic coupling between the intermediary bacteriopheophytin acceptor (I^?) and Q^?_A. It is argued that the anisotropy is too strong to arise solely from an anisotropy in the exchange interaction between I^? and Q^?_A and that dipolar contributions to the magnetic coupling between I^? and Q^?_A are important. The anisotropy in the magnetic coupling for reaction centers of Rps. sphaeroides strains 2.4.1 and R-26 is different from that of R. rubrum wild type. The combination of the 4-fold higher resolution at Q-band and the line narrowing upon deuteration has enabled us to obtain the principal g values and two hyperfine interaction constants of the reduced first quinone acceptor Q^?_A. The principal g values are g_x = 2.0067, g_y = 2.0056 and g_z = 2.0024; the hyperfine constant of the CH₂ group at position 1 is 1.6 G and that of the CH₃ group at position 2 is 2.1 G. These values are close to those found for ubisemiquinone in vitro (Okamura, M.Y., Debus, R.J., Isaacson, R.A. and Feher, G. (1980) Fed. Proc. 39, 1802; Hales, B.J. (1975) J. Am. Chem. Soc. 97, 5993–5997).     

Studies on the Redox Characteristics of Ferrioxamine E

Thermodynamic parameters for the reduction of ferrioxamine E as calculated from redox potentials determined at four different temperatures were found to be ΔH ^≠ =7.1±3.4 kJ mol^?1 and ΔS^≠=?146 J mol^?1 K^?1. The negative entropy value is large, because the decrease in the charge at the metal center and an increase in its ionic radius force the structure of the complex to become less rigid and resemble the desferrisiderophore. The hydrophilic groups of the system are now (relatively more) available for solvent interaction. Thus, a large negative entropy change accompanies the reduction of the complex. Kinetics of reduction of ferrioxamine by V^II, Cr^II, Eu^II, and dithionite were measured at different temperatures and by dithionite at different pH values. The Cr^II and Eu^II reactions proceed by an inner‐sphere mechanism and have second‐order rate constants at 25° of 1.37×10⁴ and 1.23×10⁵ M ^?1 s^?1, respectively. For the V^II reduction, the corresponding rate constant was 1.89×10³ M ^?1 s^?1. The activation parameters for the V^II reduction were ΔH^≠ = 8.3 kJ mol^?1; ΔS^≠ = ?154 J mol^?1 K^?1. These values are indicative of an outer‐sphere mechanism for V^II reduction. The reduction by dithionite is half order in dithionite concentration indicating that SO ^. is the sole reducing species. log of reduction rate constants of different trihydroxamates by this reductant were correlated with their respective redox potentials, and the variation was found to be in approximate correspondence with the expectations of Marcus relationship.     

Variable chlorophyll a fluorescence from P-700 enriched Photosystem I particles dependent on the redox state of the reaction centre

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence.2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs.3. EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite.4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles.5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P⁺X are more effective quenchers of chlorophyll fluorescence than PX^?, where P is P-700.     

Characterization of Nuclear Mutants of Maize Which Lack the Cytochrome f/b-563 Complex

Two high fluorescent, nuclear recessive mutants of maize (Zea mays L.), designated hcf-2 and hcf-6, are described which are missing the chloroplast cytochrome f/b-563 complex. Thylakoids from the mutants show a block in whole chain electron transport activity (H₂O to methyl viologen), while retaining activities associated with photosystem II (H₂O to phenylenediamine) and photosystem I (diaminodurene to methyl viologen). Chemically induced, optical difference spectra indicate a loss of cytochromes f and b-563. Cytochrome b-559 is present in both high and low potential forms. EPR analyses of thylakoid membranes of hcf-6 reveals the lack of a signal (g = 1.90) associated with the Rieske Fe-S center. Additionally, hcf-6 is lacking EPR signals at g = 6 (attributable to the high spin ferric heme of cytochrome b-563) and g = 2.5 (unidentified). The mutant retains signals at g = 2.9 (cytochrome b-559) and at g = 4.3 and 9 (both signals probably arising from a storage form of ferric iron).

Thylakoid polypeptides are examined using polyacrylamide gel electrophoresis. hcf-2 and hcf-6 have identical profiles, showing losses of polypeptides with apparent molecular masses of 33 (cytochrome f), 23 (cytochrome b-563), and 17.5 kilodaltons. The protein associated with the Rieske Fe-S center could not be determined from the gel profiles. Additionally, both mutants show an increase in a band with a molecular mass of 31 kilodaltons.