Unambiguous NOE assignments in proteins by a combination of through-bond and through-space correlations

Heteronuclear editing has found widespread use in the detection ofproton–proton dipolar interactions in isotopically labelled proteins.However, in cases where both the resonances of protons and directly bound¹³C or ¹⁵N spins of two or more sites aredegenerate, unambiguous assignments are difficult to obtain by conventionalmethods. Here, we present simple extensions of well-known triple-resonancepulse sequences which improve the dispersion of NOESY spectra. In order torecord the chemical shifts of backbone nuclei which allow a resolution ofoverlapping cross peaks, the magnetization is relayed via the scalarcoupling network either before or after the NOE mixing period. The novelpulse sequences are applied to flavodoxin from the sulfate-reducing organismDesulfovibrio vulgaris. A number of previously unassigned NOE interactionsinvolving -, - and amide protons can be unequivocallyidentified, suggesting that the accuracy of protein structure determinationcan be improved.     

Rebuilding flavodoxin from C alpha coordinates: a test study

The tertiary structure of flavodoxin has been model built from only the X-ray crystallographic alpha-carbon coordinates. Main-chain atoms were generated from a dictionary of backbone structures. Side-chain conformations were initially set according to observed statistical distributions, clashes were resolved with reference to other knowledge-based parameters, and finally, energy minimization was applied. The RMSD of the model was 1.7 A across all atoms to the native structure. Regular secondary structural elements were modeled more accurately than other regions. About 40% of the chi 1 torsional angles were modeled correctly. Packing of side chains in the core was energetically stable but diverged significantly from the native structure in some regions. The modeling of protein structures is increasing in popularity but relatively few checks have been applied to determine the accuracy of the approach. In this work a variety of parameters have been examined. It was found that close contacts, and hydrogen-bonding patterns could identify poorly packed residues. These tests, however, did not indicate which residues had a conformation different from the native structure or how to move such residues to bring them into agreement. To assist in the modeling of interacting side chains a database of known interactions has been prepared.     

Pyruvate: ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition,catalytic properties,and sequence alignments

Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon. In this communication we describe the purification and properties of pyruvate: ferredoxin oxidoreductase from this organism. The catabolic enzyme was purified 250-fold to apparent homogeneity with a yield of 16%. The native enzyme had an apparent molecular mass of 120 kDa and was composed of four different subunits of apparent molecular masses of 45, 33, 25, and 13 kDa, indicating and structure. Per mol, the enzyme contained 0.8 mol thiamine pyrophosphate, 9 mol non-heme iron, and 8 mol acid-labile sulfur. FAD, FMN, lipoic acid, and copper were not found. The purified enzyme showed an apparent K _m for coenzyme A of 0.02 mM, for pyruvate of 0.3 mM, and for clostridial ferredoxin of 0.01 mM, an apparent V _max of 64 U/mg (at 65°C) with a pH optimum near 7.5 and an Arrhenius activation energy of 75 kJ/mol (between 30 and 70°C). The temperature optimum was above 90°C. At 90°C, the enzyme lost 50% activity within 60 min in the presence of 2 M KCl. The enzyme did not catalyze the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, and hydroxypyruvate. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the -subunit had similarities to the N-terminal amino acid sequence of the -subunit of the heterotetrameric pyruvate: ferredoxin oxidoreductase from Pyrococcus furiosus and from Thermotoga maritima, and unexpectedly, to the N-terminal amino acid sequence of the homodimeric pyruvate: ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the -subunits of the enzyme from A. fulgidus and the heterodimeric pyruvate: ferredoxin oxidoreductase from Halobacterium halobium.Abbreviations CoASH Coenzyme A - F ₄₂₀ Coenzyme F₄₂₀     

A new triple-resonance experiment for the sequential assignment of backbone resonances in proteins

Summary A new protocol is described for obtaining intraresidual and sequential correlations between carbonyl carbons and amide ¹H and ¹⁵N resonances of amino acids. Frequency labeling of ¹³CO spins occurs during a period required for the ¹³C-¹⁵N polarization transfer, leading to an optimized transfer efficiency. In a four-dimensional version of the experiment, ¹³C chemical shifts are used to improve the dispersion of signals. The resonance frequencies of all backbone nuclei can be detected in a 3D variant in which cross peaks are split along two frequency axes. This pulse scheme is the equivalent of a five-dimensional experiment. The novel pulse sequences are applied to flavodoxin from Desulfovibrio vulgaris.     

Interaction of flavodoxin with cyanobacterial thylakoids

Flavodoxin from the cyanobacterium Anabaena PCC 7119 has been shown to mediate, under illumination, the transfer of electrons from the thylakoidal membranes that were isolated from the same organism, to both the enzyme ferredoxin-NADP⁺ reductase and cytochrome c. Chemical cross-linking of ferredoxin or flavodoxin to the photosynthetic membranes provides a preparation that is active in cytochrome c photoreduction without the addition of external protein carrier. NADP⁺ photoreduction, albeit diminished, was observed only after addition of exogenous electron carrier protein. Immunoblotting analysis of the chemical adduct reveals that flavodoxin binds to a 10 kDa polypeptide subunit in the cyanobacterial Photosystem I which appears to act as its physiological partner in the electron transfer process.Abbreviations Fd ferredoxin - Fld flavodoxin - cyt c cytochrome c - EDC 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide - PS I Photosystem I     

The role of iron in phytoplankton photosynthesis,and the potential for iron-limitation of primary productivity in the sea

Iron supply has been suggested to influence phytoplankton biomass, growth rate and species composition, as well as primary productivity in both high and low NO₃ ^– surface waters. Recent investigations in the equatorial Pacific suggest that no single factor regulates primary productivity. Rather, an interplay of bottom-up (i.e., ecophysiological) and top-down (i.e., ecological) factors appear to control species composition and growth rates. One goal of biological oceanography is to isolate the effects of single factors from this multiplicity of interactions, and to identify the factors with a disproportionate impact. Unfortunately, our tools, with several notable exceptions, have been largely inadequate to the task. In particular, the standard technique of nutrient addition bioassays cannot be undertaken without introducing artifacts. These so-called bottle effects include reducing turbulence, isolating the enclosed sample from nutrient resupply and grazing, trapping the isolated sample at a fixed position within the water column and thus removing it from vertical movement through a light gradient, and exposing the sample to potentially stimulatory or inhibitory substances on the enclosure walls. The problem faced by all users of enrichment experiments is to separate the effects of controlled nutrient additions from uncontrolled changes in other environmental and ecological factors. To overcome these limitations, oceanographers have sought physiological or molecular indices to diagnose nutrient limitation in natural samples. These indices are often based on reductions in the abundance of photosynthetic and other catalysts, or on changes in the efficiency of these catalysts. Reductions in photosynthetic efficiency often accompany nutrient limitation either because of accumulation of damage, or impairment of the ability to synthesize fully functional macromolecular assemblages. Many catalysts involved in electron transfer and reductive biosyntheses contain iron, and the abundances of most of these catalysts decline under iron-limited conditions. Reductions of ferredoxin or cytochrome f content, nitrate assimilation rates, and dinitrogen fixation rates are amongst the diagnostics that have been used to infer iron limitation in some marine systems. An alternative approach to diagnosing iron-limitation uses molecules whose abundance increases in response to iron-limitation. These include cell surface iron-transport proteins, and the electron transfer protein flavodoxin which replaces the Fe-S protein ferredoxin in many Fe-deficient algae and cyanobacteria.     

Molecular mechanism of metabolic NAD(P)H-dependent electron-transfer systems: The role of redox cofactors

NAD(P)H-dependent electron-transfer (ET) systems require three functional components: a flavin-containing NAD(P)H-dehydrogenase, one-electron carrier and metal-containing redox center. In principle, these ET systems consist of one-, two- and three-components, and the electron flux from pyridine nucleotide cofactors, NADPH or NADH to final electron acceptor follows a linear pathway: NAD(P)H?→?flavin?→?one-electron carrier?→?metal containing redox center. In each step ET is primarily controlled by one- and two-electron midpoint reduction potentials of protein-bound redox cofactors in which the redox-linked conformational changes during the catalytic cycle are required for the domain-domain interactions. These interactions play an effective ET reactions in the multi-component ET systems. The microsomal and mitochondrial cytochrome P450 (cyt P450) ET systems, nitric oxide synthase (NOS) isozymes, cytochrome b₅ (cyt b₅) ET systems and methionine synthase (MS) ET system include a combination of multi-domain, and their organizations display similarities as well as differences in their components. However, these ET systems are sharing of a similar mechanism. More recent structural information obtained by X-ray and cryo-electron microscopy (cryo-EM) analysis provides more detail for the mechanisms associated with multi-domain ET systems. Therefore, this review summarizes the roles of redox cofactors in the metabolic ET systems on the basis of one-electron redox potentials. In final Section, evolutionary aspects of NAD(P)H-dependent multi-domain ET systems will be discussed.     

Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein-protein interaction surface

Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP⁺ reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.     

Structure of the oxidized long-chain flavodoxin from Anabaena 7120 at 2 A resolution.

The structure of the long-chain flavodoxin from the photosynthetic cyanobacterium Anabaena 7120 has been determined at 2 A resolution by the molecular replacement method using the atomic coordinates of the long-chain flavodoxin from Anacystis nidulans. The structure of a third long-chain flavodoxin from Chondrus crispus has recently been reported. Crystals of oxidized A. 7120 flavodoxin belong to the monoclinic space group P2(1) with a = 48.0, b = 32.0, c = 51.6 A, and beta = 92 degrees, and one molecule in the asymmetric unit. The 2 A intensity data were collected with oscillation films at the CHESS synchrotron source and processed to yield 9,795 independent intensities with Rmerg of 0.07. Of these, 8,493 reflections had I > 2 sigma and were used in the analysis. The model obtained by molecular replacement was initially refined by simulated annealing using the XPLOR program. Repeated refitting into omit maps and several rounds of conjugate gradient refinement led to an R-value of 0.185 for a model containing atoms for protein residues 2-169, flavin mononucleotide (FMN), and 104 solvent molecules. The FMN shows many interactions with the protein with the isoalloxazine ring, ribityl sugar, and the 5'-phosphate. The flavin ring has its pyrimidine end buried into the protein, and the functional dimethyl benzene edge is accessible to solvent. The FMN interactions in all three long-chain structures are similar except for the O4' of the ribityl chain, which interacts with the hydroxyl group of Thr 88 side chain in A. 7120, while with a water molecule in the other two. The phosphate group interacts with the atoms of the 9-15 loop as well as with NE1 of Trp 57. The N5 atom of flavin interacts with the amide NH of Ile 59 in A. 7120, whereas in A. nidulans it interacts with the amide NH of Val 59 in a similar manner. In C. crispus flavodoxin, N5 forms a hydrogen bond with the side chain hydroxyl group of the equivalent Thr 58. The hydrogen bond distances to the backbone NH groups in the first two flavodoxins are 3.6 A and 3.5 A, respectively, whereas in the third flavodoxin the distance is 3.1 A, close to the normal value. Even though the hydrogen bond distances are long in the first two cases, still they might have significant energy because their microenvironment in the protein is not accessible to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)