Horse heart ferricytochrome c: conformation and heme configuration of low ionic strength acidic forms

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochromec in thepH range 6–0.8 and at the lowest possible ionic strengths, in water, and at 30°C are reported. The neutralpH form, state III, changes to the acidicpH form, state I, through a three-step process: state III state IIIa state II state I, with pKa's of 3.6±0.3, 2.7±0.2, and 1.2±0.2, depending on the monitoring probe, respectively. State IIIa ferricytochromec is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochromec is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 Å.     

Horse heart ferricytochromec: Conformation and heme configuration of high ionic strength acidic forms

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s ?state III _s,a ?state II _s ?state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a →state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump

A dual-wavelength fluorimeter was constructed, which used two light emitting diodes (LEDs) to excite the fluorescence dye RH 421 alternately with two different wavelengths. The ratio of the emissions at the two excitation wavelengths provided a drift-insensitive signal, which allowed detection of very small changes of the fluorescence intensity. Those small changes were induced by ion binding and release in conformation E₁ of the Na,K-ATPase. Titration experiments were performed to determine equilibrium dissociation constants (± standard deviation) for each step in the complete binding and release sequence: 0.12 ± 0.01 mM (E₂(K₂) KE₁), 0.08 ± 0.01 mM (KE₁ E₁), 3.0 ± 0.2 mM (NaE₁ E₁), 5.2 ± 0.4 mM (Na₂E₁ NaE₁) and 6.5 ± 0.4 m_M (Na₃E₁ Na₂E₁) at pH 7.2 and T=16°C. These numbers show that the affinities of the binding sites exposed to the cytoplasm, are higher for K⁺ than for Na⁺ ions, similar to what was found on the extracellular side. The physiological requirement for extrusion of Na⁺ from the cytoplasm, and for import of K⁺ from the extracellular medium seems to be facilitated not by favorable binding affinities in state E₁ but by the two ATP-driven reaction steps of the cycle, E₂(K₂) + ATP K₂E₁ · ATP and Na₃E₁ · ATP (Na₃) E_l-P, which border the ion exchange reactions at the binding sites in conformation E₁. Correspondence to: H.-J. Apell     

Mutational specificity of a conditional Escherichia coli mutator, mutD5

Summary MutD5, a conditional mutator in Escherichia coli, causes the stimulation of mutation frequencies 50 to 100 fold in minimal medium. In rich medium mutation frequencies are further increased 50 to 100 fold. We show here that all possible base-pair mutations are increased in a mutD5 strain grown in rich medium. A:TG:C transitions as well as A:TC:G, A:TT:A aud G:CC:G transversions are stimulated. Transitions occur more frequently than transversions. MutD5 also increases the reversion frequencies of three trpA frameshift mutations by causing base-pair additions, and, possibly, base-pair deletions.     

Diverse polymorphism within a short coding region of the human aldehyde dehydrogenase-5 (ALDH5) gene

Human aldehyde dehydrogenase-5 gene (originally named as ALDHX) is expressed in liver and testis. The ALDH5 does not contain introns in the coding sequence for 517 amino acid residues. Within a short nucleotide region of the gene, the following three nucleotide changes were found in high frequencies, i.e., a silent CT at nucleotide (nt) 183, CT at nt 257 associated with a ValAla substitution, and TG at nt 320 associated with a ArgLeu substitution. The frequency of C at nt 183 is 81% in Caucasians and 65% in Japanese, and the difference is statistically not significant. The frequency of C at nt 257 is 76% in Caucasians and 55% in Japanese, and the difference is statistically significant (P = 0.02). The frequency of T at nt 320 is 71% in Caucasians, while it is only 27% in Japanese. The racial difference at nt 320 is highly significant (P < 0.001). No significant difference was found in the genotypes of the three nucleotide positions between alcoholic and nonalcoholic Caucasians within the limited numbers of subjects examined.     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

The continuum of pure and hybrid myosin heavy chain-based fibre types in rat skeletal muscle

The myosin heavy chain (MHC)-based fibre composition of adult rat adductor magnus (AM) and tibialis anterior (TA) muscles was investigated using single fibre analysis. Microelectrophoresis performed on single fibre fragments demonstrated a predominance of pure fast MHC-based fibre types (expressing only one fast MHC). Most of the fibres analysed from both the AM (72%) and TA (50%) were pure type IIB (expressing only MHCIIb). Pure type IID fibres (expressing only MHCIId) were also abundant in AM (20%) and TA (18%). In addition, hybrid fibres coexpressing MHCIIb and MHCIId in varying proportions (fibre types IIBD and IIDB) were found, as well as fibres coexpressing MHCIId and MHCIIa with a predominance of MHCIId (type IIDA) and some C fibres (coexpressing MHCI and MHCIIa in varying proportions). Considered altogether, these data reflect the dynamic nature of adult skeletal muscle fibres and indicate a continuum of MHC-based fibre types in normal rat muscle with transitions in the order IIB IIBD IIDB IID IIDA IIAD IIA IIC IC I.     

DNA-dependent RNA polymerases from Schneider 2-L cells of Drosophila. I. Preliminary characterization

The DNA-dependent RNA polymerases of Schneider 2-L cells of Drosophila melanogaster are described. These cells contain five readily detectable forms of this enzyme, polymerases I_a, I_b, III_a, II, and III_b, which elute from DEAE-Sephadex at 0.08, 0.12, 0.15, 0.20, and 0.22 m ammonium sulfate, respectively. RNA polymerases III_a and III_b, which each constitute about 5–10% of the total RNA polymerase activity in Drosophila embryos, are found to constitute 30 and 10%, respectively, of the total polymerase activity in cultured cells. The two form III polymerases are further characterized by in vitro response to divalent cations and ionic strength, template utilization, and sensitivity to -amanitin. Verification of the class III designation of these two polymerases is provided by their sensitivity to only very high levels of -amanitin (50% inhibition at approximately 800 µg/ml), their 10-fold greater activity on poly[d(A–T)], and their elution from DEAE-cellulose at lower ionic strengths than from DEAE-Sephadex.This work was supported by the Natural Sciences and Engineering Research Council.     

The ‘A’ genome donor of Eleusine coracana (L.) Gaertn. (Gramineae)

Summary In an attempt to discover A and B genome donor(s) to finger millet, Eleusine coracana, or its progenitor species, E. africana (both allotetraploid 2n=4x=36), five diploid species, E. Indica, E. Floccifolia, E. multiflora, E. tristachya and E. intermedia, were crossed to finger millet and its progenitor taxon. Crosses were successful only with E. coracana. Three combinations of triploid hybrids E. coracana x E. indica, E. coracana x E. floccifolia, and E. coracana x E. multiflora were obtained and analysed. Meiotic behaviour was perfectly normal in parental species. The regular number of 18 bivalents in E. coracana, 9 bivalents in E. indica, E. intermedia, E. tristachya and E. floccifolia and 8 bivalents in E. multiflora were invariably noticed. In E. coracana x E. indica hybrids a mean chromosome pairing of 8.84_I+8.80_II+0.03_III+0.10_IV per cell was found. About 86.5% of the cells showed the typical 9_I+9_II configuration, suggesting that E. indica (AA) is one of the diploid genome donors to cultivated species E. coracana. A mean chromosome pairing of 11.08_I+7.63_II+0.16_III+0.04_IV per cell was found in E. coracana x E. floccifolia hybrids. Two to ten bivalents and varying numbers of univalents were seen in 55% of the cells. About 45% of the cells showed the 9_I+9_II configuration. Various evidence suggests that perennial E. floccifolia is a primitive member of the A genome group of Eleusine species, and it may not be a genome donor to E. coracana. In E. coracana x E. multiflora hybrids (2n=26) mean chromosome pairing of 21.45_I+1.97_II+0.13_III+0.04_IV per cell was found. About 91% of the cells were observed to have 20–26 univalents. Only a small percentage of the cells contained bivalents or multivalents. This pairing behaviour indicates that E. multiflora lacks genomic homology with the A or B genome of E. coracana. Genomically E. multiflora is a distinct species and a genomic symbol of C is assigned to it. Identification of the B genome donor species to cultivated millet. E. coracana remains elusive.     

Plant distribution in relation to the length of the growing season in a snow-bed in the Taisetsu Mountains,northern Japan

The distribution pattern of plants was studied in an alpine snow-bed in six plots along a snow-melting gradient. Each plot consisted of two habitats with respect to the microtopography; the flat habitat and the mound habitat. The number of species per plot decreased with the shortened snow-free period. In the flat habitat, the dominant growth forms changed from the early exposed plots to the late exposed ones as follows; lichens evergreen and deciduous shrubs forbs graminoids bryophytes. In the mound habitat, evergreen and deciduous shrubs prevailed widely along the gradient because of the ability to exploit new habitat by creeping over exposed rocks. For shrubs, the existence of mounds contributed to the expansion of the distribution ranges. Forbs and graminoids shifted their distribution modes to the late exposed plots where shrubs decreased in cover. Deciduous shrubs and forbs completely disappeared in the latest exposed plot.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

Comparative Field Summer Stress of Three Tree Species Co-occurring in Mediterranean Coastal Dunes

Chlorophyll a fluorescence, water potential (_s), and root system of Juniperus oxycedrus ssp. macrocarpa, Juniperus phoenicea ssp. turbinata, and Pinus pinea were studied in Mediterranean coastal dunes of SW Spain during summer drought and after fall rains in 1999, the driest year in the 90's. A strong and reversible depression in the photochemical efficiency of photosystem 2 of the three species was recorded, which happened concomitantly with the diurnal increase and decrease in radiation. J. phoenicea, with superficial root system, was the most affected species by summer drought. It showed high rates of down-regulation of photosynthesis by photoinhibition and positive correlation between _s and F_v/F_p, with _s lower than -7 MPa. However, it tolerated this high stress, showing a fast recovery of its physiological state after fall rains. On the other hand, J. oxycedrus and P. pinea, both with deep root systems, kept their _s values up to -3 MPa, showing lower stress during summer drought. On the other hand, J. oxycedrus and J. phoenicea were more sensible to changes in edaphic water content than P. pinea. These specific responses to summer drought would be determined by their root distributions and stomatal control of transpiration, conditioning the efficiency in getting and using the available water resources. Ecophysiological responses indicate that these species are well-adapted to long periods of drought in Mediterranean climate areas, developing different strategies: J. phoenicea tolerates high stress with a fast recovery after fall rains, while J. oxycedrus and P. pinea are less affected by summer drought since their deep root systems would allow them to reach deep water resources.     

Influence of Phosphorus Application on Water Relations, Biochemical Parameters and Gum Content in Cluster Bean Under Water Deficit

Relative water content (RWC), leaf water potential (_w) and osmotic potential (_s), contents of chlorophyll (Chl) a, Chl b, soluble sugars, and seed quality (gum content) were used to evaluate the role of phosphorus in alleviation of the deleterious effect of water deficit in clusterbean (Cyamopsis tetragonoloba L. Taub). Under water stress, _w, _s, and Chl and gum contents decreased and soluble sugar contents increased. Phosphorus application increased Chl and sugar contents in control plants and ameliorated negative effects of water stress.     

Enhancement of oxygen mass transfer in stirred bioreactors using oxygen-vectors. 1. Simulated fermentation broths

Oxygen mass transfer represents the most important parameter involved in the design and operation of mixing-sparging equipment for bioreactors. It can be described and analyzed by means of the mass transfer coefficient, k_La. The k_La values are affected by many factors such as geometrical and operational characteristics of the vessels, media composition, type, concentration and microorganism morphology, and biocatalysts properties. The efficiency of oxygen transfer could be enhanced by adding oxygen-vectors in broths, such as hydrocarbons or fluorocarbons, without increasing the energy consumption for mixing or aeration. The experimental results obtained for simulated broths indicated a considerable increase of k_La in the presence of n-dodecane, and the existence of a certain value of n-dodecane concentration that corresponds to a maximum mass transfer rate of oxygen. The magnitude of the positive effect of n-dodecane depends both on the broths characteristics and operational conditions of the bioreactor.Notation d stirrer diameter, mm - d oxygen electrode diameter, mm - D bioreactor diameter, mm - h distance from the inferior stirrer to the bioreactor bottom, mm - H bioreactor height, mm - k_La oxygen mass transfer coefficient, s^-1 - l impeller blade length, mm - I oxygen electrode immersed length, mm - P power consumption for mixing of non-aerated broths, W - P_a power consumption for mixing of aerated broths, W - (P_a/V) specific power input, W/m³ - s baffle width, mm - v_S superficial air velocity, m/s - V volume of medium, m³ - w impeller blade height, mm - volumetric fraction of oxygen-vector - _a apparent viscosity, Pa*s - density, kg/m³     

Active-site sulfhydryl chemistry plays a major role in the misfolding of urea-denatured rhodanese

Unfolded bovine rhodanese, a sulfurtransferase, does not regain full activity upon refolding due to the formation of aggregates and disulfide-linked misfolded states unless a large excess of reductant such as 200 mM -ME and 5 mg/ml detergent are present [Tandon and Horowitz (1990), J. Biol. Chem. 265, 5967]. Even then, refolding is incomplete. We have studied the unfolding and refolding of three rhodanese forms whose crystal structures are known: ES, containing the transferred sulfur as a persulfide; E, without the transferred sulfur, and carboxymethylated rhodanese (CMR), in which the active site was blocked by chemical modification. The X-ray structures of ES, E, and CMR are virtually the same, but their tertiary structures in solution differ somewhat as revealed by near-UV CD. Among these three, CMR is the only form of rhodanese that folds reversibly, requiring 1 mM DTT. A minimum three-state folding model of CMR (NIU) followed by fluorescence at 363 nm, (NI) by fluorescence at 318 nm, and CD (IU) is consistent with the presence of a thermodynamically stable molten globule intermediate in 5–6 M urea. We conclude that the active-site sulfhydryl group in the persulfide form is very reactive; therefore, its modification leads to the successful refolding of urea-denatured rhodanese even in the absence of a large excess of reductant and detergent. The requirement for DTT for complete reversibility of CMR suggests that oxidation among the three non-active-site SH groups can represent a minor trap for refolding through species that can be easily reduced.     

Novel strategies for sensitivity enhancement in heteronuclear multi—dimensional NMR experiments employing pulsed field gradients

Summary Novel strategies for sensitivity enhancement in heteronuclear multidimensional spectra are introduced and evaluated theoretically and experimentally. It is shown that in 3D sequences employing several Coherence Order Selective Coherence Transfer (COS-CT) steps, enhancement factors of up to 2 can be achieved. This sensitivity enhancement is compatible with the use of heteronuclear gradient echoes, yielding spectra with excellent water suppression. HNCO and HCCH-TOCSY pulse sequences are proposed and experimentally tested. These experiments employ recently developed coherence order selective pulse sequence elements, e.g., COS-INEPT and planar TOCSY for antiphase to in-phase transfers 2F^-S₂S^- or in-phaseaCOS-CT for in-phase transfer F^-S^-, and the well-known isotropic TOCSY mixing sequences for homo- and heteronuclear in-phase transfer.This work has been presented in part at the 35th ENC, April 10–15, 1994, Asilomar, CA, U.S.A.     

Structure of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex: new prosthetic groups,Q-space,and the ‘hors d’oeuvres hypothesis’ for assembly of the complex

3-Å crystal structures of the cytochrome b₆f complex have provided a structural framework for the photosynthetic electron transport chain. The structures of the 220,000 molecular weight dimeric cytochrome b₆f complex from the thermophilic cyanobacterium, Mastigocladis laminosus (Kurisu et al. 2003, Science 302: 1009–1014), and the green alga, Chlamydomonas reinhardtii (Stroebel et al. 2003, Nature 426: 413–418), are very similar. The latter is the first structure of a integral membrane photosynthetic electron transport complex from a eukaryotic source. The M. laminosus and C. reinhardtii structures have provided structural information and experimental insights to the properties and functions of three native and novel prosthetic groups, a chlorophyll a, a -carotene, and a unique heme x, one copy of which is found in each monomer of the cytochrome b₆f complex, but not the cytochrome bc₁ complex from the mitochondrial respiratory chain of animals and yeast. Several functional insights have emerged from the structures including the function of the dimer; the properties of heme x; the function of the inter-monomer quinone-exchange cavity; a quinone diffusion pathway through relatively narrow crevices or portals; a modified reaction scheme for n-side quinone redox reactions; a necessarily novel mechanism for quenching of the bound chlorophyll triplet state; a possible role for the bound chlorophyll a in activation of the LHC kinase; and a structural and assembly role for the four small PetG, L, M, and N subunits. An hors doeuvres hypothesis for assembly of the complex is proposed for the small hydrophobic stick or picket fence polypeptides at the periphery of the complex, based on the cis-positive orientation of the small hydrophobic subunits and the toothpick binding mode of the -carotene.     

Inheritance and linkage relationships of glutamate oxaloacetate transaminase isoenzymes in apple

Summary Four zones of enzymatic activity for glutamate oxaloacetate transaminase (GOT) were found in apple tissue. A dimeric gene, GOT-1, determining the fastest migrating zone, was identified. Six alleles were found, including a near null allelle which produced detectable heterodimeric bands but not homodimeric bands. A marked deficit or absence of certain geno-types in all backcrosses and in some crosses between unrelated varieties was attributed to the close linkage (r=0.02±0.005) of GOT-1 with the incompatibility S locus. GOT-1 was also closely linked with the isocitrate dehydrogenase locus IDH-1 (0.03±0.01). Proposed incompatibility genotypes for four cultivars, and the linked GOT-1 alleles are Cox: S ₁ b/S ₂ d, Idared: S ₃ a/S ₄ c, Fiesta: S ₃ a/S ₂ d and Kent: S ₃ a/S ₁ b.The results reported in this paper are part of a PhD Thesis by the first author     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.