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A "minispectrin" has been constructed from the tail end of the alpha/beta heterodimer, and its actin-binding properties have been characterised. It is a complex of the N-terminal fragment of the beta-subunit consisting of the actin-binding domain plus the two first triple-helical repeats beta 1 and beta 2, and the C-terminal fragment of the alpha-subunit containing the repeats alpha 19 and alpha 20 plus the calmodulin-like domain. This minispectrin exists in a dimeric form that contains one copy of each polypeptide and binds to actin in a cooperative manner with an apparent K(d) of 2.5 microM. Calcium seems not to have any effect on its binding to actin. Electron microscopic analysis shows that the minispectrin decorates actin filaments as clusters, and induces formation of actin bundles. This study shows that the actin-binding region of the spectrin alpha/beta heterodimer retains its functional properties in a truncated form and establishes basis for further research on spectrin's structure and function.

Bacterial nitric oxide reductases are integral membrane proteins that catalyze the reduction of two molecules of nitric oxide to nitrous oxide and water. They are diverged members of the superfamily of heme/copper oxidases. The enzyme from Paracoccus denitrificans (NorBC) contains two subunits; NorB comprises the membrane-integrated active site, which harbors a heme iron/non-heme iron dinuclear center. NorC is a membrane-anchored c-type cytochrome and presumably the site of electron uptake. A DNA construct encoding the water-soluble domain of NorC (NorC(sol)) was coexpressed with the cytochrome c maturation genes in Escherichia coli. Using redox potentiometry, electronic absorption, circular dichroism (CD), magnetic CD (MCD), nuclear magnetic resonance, and electron paramagnetic resonance (EPR) spectroscopy the following observations were made: (i) NorC(sol) was folded into a alpha-helical structure. (ii) The low-spin heme iron was coordinated by histidine and methionine in both redox states. (iii) The midpoint redox potential of the NorC(sol) heme was 183 mV, much lower than the corresponding value of 275 mV in the NorBC complex. This points to an increased solvent exposure of the NorC(sol) heme compared to in the native NorBC complex and shows that the electronic properties of NorC are modulated by NorB in the complex. (iv) The EPR and MCD spectra of NorC(sol) were considered alongside the spectra of NorBC, which has helped to resolve the contribution that different redox centers make in the holo-enzyme complex.

International audience; Spectrin repeats are triple-helical coiled-coil domains found in many proteins that are regularly subjected to mechanical stress. We used atomic force microscopy technique and steered molecular dynamics simulations to study the behavior of a wild-type spectrin repeat and two mutants. The experiments indicate that spectrin repeats can form stable unfolding intermediates when subjected to external forces. In the simulations the unfolding proceeded via a variety of pathways. Stable intermediates were associated to kinking of the central helix close to a proline residue. A mutant stabilizing the central helix showed no intermediates in experiments, in agreement with simulation. Spectrin repeats may thus function as elastic elements, extendable to intermediate states at various lengths.

We have applied resonance Raman spectroscopy to investigate the properties of the dinuclear center of oxidized, reduced, and NO-bound nitric-oxide reductase from Paracoccus denitrificans. The spectra of the oxidized enzyme show two distinct νas(Fe-O-Fe) modes at 815 and 833 cm−1 of the heme/non-heme diiron center. The splitting of the Fe-O-Fe mode suggests that two different conformations (open and closed) are present in the catalytic site of the enzyme. We find evidence from deuterium exchange experiments that in the dominant conformation (833 cm−1mode, closed), the Fe-O-Fe unit is hydrogen-bonded to distal residue(s). The ferric nitrosyl complex of nitric-oxide reductase exhibits the ν(Fe3+-NO) and ν(N-O) at 594 and 1904 cm−1, respectively. The nitrosyl species we detect is photolabile and can be photolyzed to generate a new form of oxidized enzyme in which the proximal histidine is ligated to hemeb 3, in contrast to the resting form. Photodissociation of the NO ligand yields a five-coordinate high-spin heme b 3. Based on the findings reported here, the structure and properties of the dinuclear center of nitric- oxide reductase in the oxidized, reduced, and NO-bound form as well as its photoproduct can be described with certainty.

The cytochrome cbb3 is an isoenzyme in the family of cytochrome c oxidases. This protein purified from Pseudomonas stutzeri displays a cyanide-sensitive nitric oxide reductase activity (Vmax=100+/-9 mol NO x mol cbb3(-1) x min(-1) and Km=12+/-2.5 microm), which is lost upon denaturation. This enzyme is only partially reduced by ascorbate, and readily re-oxidized by NO under anaerobic conditions at a rate consistent with the turnover number for NO consumption. As shown by transient spectroscopy experiments and singular value decomposition (SVD) analysis, these results suggest that the cbb3-type cytochromes, sharing structural features with bacterial nitric oxide reductases, are the enzymes retaining the highest NO reductase activity within the heme-copper oxidase superfamily.

Background: α-Actinin is a ubiquitously expressed protein found in numerous actin structures. It consists of an N-terminal actin binding domain, a central rod domain, and a C-terminal domain and functions as a homodimer to cross-link actin filaments. The rod domain determines the distance between cross-linked actin filaments and also serves as an interaction site for several cytoskeletal and signaling proteins. Results: We report here the crystal structure of the α-actinin rod. The structure is a twisted antiparallel dimer that contains a conserved acidic surface. Conclusions: The novel features revealed by the structure allow prediction of the orientation of parallel and antiparallel cross-linked actin filaments in relation to α-actinin. The conserved acidic surface is a possible interaction site for several cytoplasmic tails of transmembrane proteins involved in the recruitment of α-actinin to the plasma membrane.

The pH-induced conformational transition in the CuA domain of subunit II of cytochrome oxidase of Paracoccus denitrificans (PdII) has been investigated using various spectroscopic and stopped-flow kinetic methods. UV-visible absorption and circular dichroism studies showed that an increase in pH from 6 to 10 leads to a conformation change with pK(a) = 8.2 associated with the CuA site of the protein. The secondary structure of the protein was, however, shown to remain unchanged in these two conformational states. Thermal and urea-induced unfolding studies showed that the "low-pH" conformation is more stable compared to the "high-pH" conformation of the protein. Moreover, the overall stability of the protein was found to decrease on reduction of the metal centers in the low-pH form, while the oxidation state of the metal centers did not have any significant effect on the overall stability of the protein in the high-pH form. Stopped-flow pH-jump kinetic studies suggested that the conformational transition is associated with a slow deprotonation step followed by fast conformational equilibrium. The results are discussed in the light of understanding the pH-induced conformational change in the beta-barrel structure of the protein and its effect on the coordination geometry of the metal site.

Nitrous-oxide reductases (N2OR) catalyze the two-electron reduction of N(2)O to N(2). The crystal structure of N2ORs from Pseudomonas nautica (Pn) and Paracoccus denitrificans (Pd) were solved at resolutions of 2.4 and 1.6 A, respectively. The Pn N2OR structure revealed that the catalytic CuZ center belongs to a new type of metal cluster in which four copper ions are liganded by seven histidine residues. A bridging oxygen moiety and two other hydroxide ligands were proposed to complete the ligation scheme (Brown, K., Tegoni, M., Prudencio, M., Pereira, A. S., Besson, S., Moura, J. J. G., Moura, I., and Cambillau, C. (2000) Nat. Struct. Biol. 7, 191-195). However, in the CuZ cluster, inorganic sulfur chemical determination and the high resolution structure of Pd N2OR identified a bridging inorganic sulfur instead of an oxygen. This result reconciles the novel CuZ cluster with the hitherto puzzling spectroscopic data.

Background: Pleckstrin homology (PH) domains constitute a structurally conserved family present in many signaling and regulatory proteins. PH domains have been shown to bind to phospholipids, and many function in membrane targeting. They generally have a strong electrostatic polarization and interact with negatively charged phospholipids via the positive pole. On the basis of electrostatic modeling, however, we have previously identified a class of PH domains with a predominantly negative charge and predicted that these domains recognize other targets. Here, we report the first experimental structure of such a PH domain. Results: The structure of the PH domain from Caenorhabditis elegans muscle protein UNC-89 has been determined by heteronuclear NMR. The domain adopts the classic PH fold, but has an unusual closed conformation of the "inositol binding loops. This creates a small opening to a deep hydrophobic pocket lined with negative charges on one side, and provides a molecular explanation for the lack of association with inositol-1,4,5-triphosphate. As predicted, the PH domain of UNC-89 has a strongly negative overall electrostatic potential. Modeling the Dbl homology (DH)-linked PH domains from the C. elegans genome shows that a large proportion of these modules are negatively charged. Conclusions: We present the first structure of a PH domain with a strong negative overall electrostatic potential. The presence of a deep pocket lined with negative charges suggests that the domain binds to ligands other than acidic phospholipids. The abundance of this class of PH domain in the C. elegans genome suggests a prominent role in mediating protein–protein interactions.

Spectrin is a vital and abundant protein of the cytoskeleton. It has an elongated structure that is made by a chain of so-called spectrin repeats. Each repeat contains three antiparallel α-helices that form a coiled-coil structure. Spectrin forms an oligomeric structure that is able to cross-link actin filaments. In red cells, the spectrin/actin meshwork underlying cell membrane is thought to be responsible for special elastic properties of the cell. In order to determine mechanical unfolding properties of the spectrin repeat, we have used single molecule force spectroscopy to study the states of unfolding of an engineered polymeric protein consisting of identical spectrin domains. We demonstrate that the unfolding of spectrin domains can occur in a stepwise fashion during stretching. The force–extension patterns exhibit features that are compatible with the existence of at least one intermediate between the folded and the completely unfolded conformation. Only those polypeptides that still contain multiple intact repeats display intermediates, indicating a stabilisation effect. Precise force spectroscopy measurements on single molecules using engineered protein constructs reveal states and transitions during the mechanical unfolding of spectrin. Single molecule force spectroscopy appears to open a new window for the analysis of transition probabilities between different conformational states.

Rhodothermus (R.) marinus, a thermohalophilic Gram-negative, and strict aerobic bacterium,has a rather distinct respiratory chain, containing a caa 3 terminal oxidase, a novel cytochromebc complex and a HiPIP, which is an electron carrier between this complex and a terminaloxidase (Pereira et al (1999a, c). To further elucidate this unusual respiratory system, itsmembrane-bound heme centers were characterized by visible and EPR spectroscopies as wellas by redox potentiometry. Rhodothermus marinus contains mostly B- and C-type hemes; asmall amount of A-type heme is also detected. The heme centers have relatively low reductionpotentials, ranging from ca. +250 to −60 mV, at pH 7. A Rieske-type center was not detected,suggesting the absence of a canonical complex III. The major terminal oxidase expressed byR. marinus is a cbb 3-type oxidase. Its presence is in agreement with molecular biology studies,which reveal the existence of a gene encoding for a FixN-type oxidase. The oxidase waspartially purified and appears to have five subunits, with apparent molecular masses of 64,57, 36, 26 (C-type heme subunit), and 13 kDa. It contains two low-spin heme C centers, onehigh-, and one low-spin heme B centers. A full description of the equilibrium redox behaviorof the heme centers was obtained for a cbb 3 oxidase for the first time. The optical spectrumfor each heme center and the corresponding reduction potentials were determined at pH 7:+ 195 (heme C), +120 (heme B), −50 (heme C), and −50 mV (heme B3).

Pleckstrin homology (PH) domains form a structurally conserved family that is associated with many regulatory pathways within the cell. Domains with a nearly identical fold are found in other families that share no sequence similarity, suggesting the existence of a stable PH superfold. The PH domains generally function as regulated membrane-binding modules that bind to inositol lipids and respond to upstream signals by targeting the host proteins to the correct cellular sites. The other domains with a similar fold, such as the phosphotyrosine binding domains, recognize protein ligands.

Background: The activity of Bruton's tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. Results: We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D- myo -inositol 1,3,4,5-tetra-kisphosphate (Ins (1,3,4,5)P 4 ). The inositol Ins (1,3,4,5)P 4 binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C- δ . A second Ins (1,3,4,5)P 4 molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P 4 to the wild type (K d = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. Conclusions: Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a G α protein.

Background: The actin-binding site of several cytoskeletal proteins is comprised of two calponin homology (CH) domains in a tandem arrangement. As a single copy, the CH domain is also found in regulatory proteins in muscle and in signal-transduction proteins. The three-dimensional structures of three CH domains are known, but they have not yet clarified the molecular details of the interaction between actin filaments and proteins harbouring CH domains. Results: We have compared the crystal structure of a CH domain from β -spectrin, which has been refined to 1.1 a resolution, with the two CH domains that constitute the actin-binding region of fimbrin. This analysis has allowed the construction of a structure-based sequence alignment of CH domains that can be used in further comparisons of members of the CH domain family. The study has also improved our understanding of the factors that determine domain architecture, and has led to discussion on the functional differences that seem to exist between subfamilies of CH domains, as regards binding to F-actin. Conclusions: Our analysis supports biochemical data that implicate a surface centered at the last helix of the N-terminal CH domain as the most probable actin-binding site in cytoskeletal proteins. It is not clear whether the C-terminal domains of the tandem arrangement or the single CH domains have this function alone. This may imply that although the CH domains are homologous and have a conserved structure, they may have evolved to perform different functions.

A novel, improved method for purification of nitric oxide reductase (NOR) from membranes of Paracoccus denitrificans has been developed. The purified enzyme is a cytochrome bc complex which, according to protein chemical and hydrodynamic data, contains two subunits in a 1:1 stoichiometry. The purified NorBC complex binds 0.87 g of dodecyl maltoside/g of protein and forms a dimer in solution. Similarly, it is dimeric in two-dimensional crystals. Images of these crystals have been processed at 8 A resolution in projection to the membrane. The NorB subunit is homologous to the main catalytic subunit of cytochrome oxidase and is predicted to contain the active bimetallic center in which two NO molecules are turned over to N2O. Metal analysis and heme composition implies that it binds two B-type hemes and a nonheme iron but no copper. NorC is a membrane-anchored cytochrome c. Fourier transform infrared spectroscopy shows that carbon monoxide dissociates from the reduced heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases. Electron paramagnetic resonance spectroscopy reveals that NO binds to the reduced enzyme under turnover conditions giving rise to signals near g = 2 and g = 4. The former represents a typical nitrosyl-ferroheme signal whereas the latter is a fingerprint of a nonheme iron/NO adduct. We conclude that the active site of NOR is a dinuclear iron center.

Nitric oxide reductase (NOR) is a key enzyme in denitrification, reforming the N-N bond (making N2O from two NO molecules) in the nitrogen cycle. It is a cytochrome bc complex which has apparently only two subunits, NorB and NorC. It contains two low-spin cytochromes (c and b), and a high-spin cytochrome b which forms a binuclear center with a non-heme iron. NorC contains the c-type heme and NorB can be predicted to bind the other metal centers. NorB is homologous to the major subunit of the heme/copper cytochrome oxidases, and NOR thus belongs to the superfamily, although it has an Fe/Fe active site rather than an Fe/Cu binuclear center and a different catalytic activity. Current evidence suggests that NOR is not a proton pump, and that the protons consumed in NO reduction are not taken from the cytoplasmic side of the membrane. Therefore, the comparison between structural and functional properties of NOR and cytochrome c- and quinol-oxidizing enzymes which function as proton pumps may help us to understand the mechanism of the latter. This review is a brief summary of the current knowledge on molecular biology, structure, and bioenergetics of NOR as a member of the oxidase superfamily.

An extensive assignment of the 1H NMR spectra of a CuA domain from the cytochrome c oxidase of Paracoccus denitrificans has been obtained on the basis of dipolar connectivities coupled to the available structural information. The assignment has been extended with the aid of 2H NMR spectra on a protein sample with cysteines selectively labeled at the β position. The spectra have been compared to those published earlier for a similar CuA fragment from the Thermus thermophilus oxidase and for a CuA construct in a blue copper protein, amicyanin. The shifts and their temperature dependence are discussed in terms of molecular orbital descriptions available for these systems. The mechanisms for the fast electron relaxation, which make the obtainment of relatively sharp NMR signals possible, are discussed. It is shown that the proton shifts of one histidine are variable from one system to the other, possibly reflecting changes in its interaction with the Cu2S2 diamond structure. Large electron delocalization onto...

Cytoskeletal proteins belonging to the spectrin family have an elongated structure composed of repetitive units. The three-dimensional solution structure of the 16th repeat from chicken brain α-spectrin (R16) has been determined by NMR spectroscopy and distance geometry-simulated annealing calculations. We used a total of 1035 distance restraints, which included 719 NOE-based values obtained by applying the ambiguous restraints for iterative assignment (ARIA) method. In addition, we performed a direct refinement against 1H-chemical shifts. The final ensemble of 20 structures shows an average RMSD of 1.52 A from the mean for the backbone atoms, excluding loops and N and C termini. R16 is made up of three antiparallel α-helices separated by two loops, and folds into a left-handed coiled-coil. The basic unit of spectrin is an antiparallel heterodimer composed of two homologous chains, β and α. These assemble a tetramer via a mechanism that relies on the completion of a single repeat by association of the partial repeats located at the C terminus of the β-chain (two helices) and at the N terminus of the α-chain (one helix). This tetramer is the assemblage able to cross-link actin filaments. Model building by homology of the “tetramerization” repeat from human erythrocyte spectrin illuminates the possible role of point mutations which cause hemolytic anemias.

The three-dimensional structure of the calponin homology domain present in many actin binding cytoskeletal and signal-transducing proteins has been determined at 2.0 A resolution.

The CuA site of cytochrome c oxidase and the type 1 Cu site of cupredoxins occur in homologous protein folds with a His2Cys ligand set, but are distinguished by the presence of a second Cys and a second Cu in CuA that result in the formation of a dithiolate-bridged dinuclear Cu cluster. The resonance Raman (RR) spectrum of the soluble CuA-containing fragment from Paracoccus denitrificans exhibits intense vibrations at 260 and 339 cm-1. Their respective 34S-isotope shifts of −4.1 and −5.1 cm-1 allow them to be assigned to two Cu−S stretching modes, ν(Cu−S), of the Cu2S2Im2 moiety. A normal coordinate analysis (NCA) of the RR spectra of CuA substituted with isotopes of S, Cu, and N was carried out to determine whether it is possible to distinguish between dinuclear models with bridging or terminal cysteine ligands. Whereas the terminal Cys model predicts that both of the ν(Cu−S) modes lie between 340 and 350 cm-1, the bridging Cys geometry successfully predicts the S-shifts at 260 and 339 cm-1. Thus, the Ra...

Cytochrome-c oxidase contains an unusual copper centre (CuA) located in subunit II. This centre mediates one-electron transfer from cytochrome c to low-spin heme a. Recent spectroscopic and biochemical studies have shown that this centre is a valence delocalised dinuclear [Cu(+1.5)-Cu(+1.5)] centre. We have measured the absorption, EPR and variable-temperature magnetic circular dichroism spectra of the CuA-binding domain isolated from Paracoccus denitrificans cytochrome aa3. The EPR spectrum showed the following signals: g‖= 2.18; g⊥= 2.03. g‖ exhibited a seven-line hyperfine splitting pattern, with an intensity ratio showing that the single unpaired electron interacted equally with two copper nuclei. The magnetic circular dichroism spectrum was identical to those from CuA in bovine heart cytochrome-c oxidase and centre A of nitrous-oxide reductase, showing the close structural similarity between the three centres. To identify the ligands of CuA, all the conserved putative ligands in the P. denitrificans CuA domain were substituted. Only five residues, Cys244, Cys248, His209, His252, and Met255, were required for correct assembly of the CuA centre. Replacement of Met255 caused protein misfolding. Hence, methionine may have a structural role for the folding of the protein rather than being a CuA ligand. Given that both copper ions must have identical coordination geometries, the number of possible structures is limited. Two models are proposed: one involves the thiolate side-chains of Cys244 and Cys248 bridging a pair of copper ions with one histidine coordinating each copper ion, and the other has terminal ligation of each copper ion by one cysteine and one histidine residue. In both models, the metal-metal distance can be sufficiently short to permit direct d-orbital overlap of the copper ions. The magnetic circular dichroism transitions at 475 nm and 525 nm are assigned to thiolate-to-copper charge-transfer processes polarised perpendicular to one another, although the magnetic circular dichroism intensities show that the excited states were heavily mixed with copper d-orbitals. These intensities can be interpreted in the thiolate bridged model in terms of transitions within a Cu2(SR)2 rhomb. In the model involving terminal cysteine ligation, exciton coupling of two thiolate-to-copper charge-transfer transitions of similar energy, polarised along the Cu-S bonds, would contribute two transitions perpendicular to one another. This requires that the cysteine ligands have a cis orientation relative to one another. The spectral properties of the H252N variant (histidine at position 252 replaced by asparagine) and the high-pH form of P. denitrificans CuA were similar, showing that one copper ion had lost one histidine ligand in the latter form. The dimer was converted into a valence trapped [Cu(+1)-Cu(+2)] state, which may retain the metal-metal interaction.

Subunit II of cytochrome c oxidase has a C-terminal domain that is exposed to aqueous solution on membrane surface and contains a copper center called CuA. The central part of the cytochrome c binding site is thought to reside in this domain. We have expressed the subunit II fragment of the Paracoccus denitrificans cytochrome c oxidase in a soluble form and studied its interaction with cytochrome c by stopped-flow spectroscopy. The oxidation of cytochrome c by the CuA domain follows monophasic kinetics, indicating the presence of a single kinetically competent binding site. In low ionic strength medium, the domain oxidizes Paracoccus cytochrome c-550 and horse mitochondrial cytochrome c at the rates of 1.5 x 10(6) and 3 x 10(5) M-1 s-1, respectively. The reaction rates are strongly dependent on ionic strength, which must reflect electrostatic interactions within the complex. The KD for the complex between the bacterial cytochrome c and the domain is 1.6 microM; i.e., it is similar to that between the mitochondrial cytochrome c and the intact oxidase, suggesting that both contain the same catalytically competent binding site. Using site-directed mutagenesis, we have identified five conserved residues of the CuA domain that are involved in the cytochrome c binding. Mutations of glutamine 148, glutamate 154, aspartate 206, aspartate 221, or glutamate 246 lead to a 35-85% decrease in the rate of cytochrome c oxidation. The simultaneous substitution of three invariant carboxylic acids (aspartate 206, aspartate 221, and glutamate 246) leads to a 95% decrease in the reaction rate. Conversely, the reaction can be enhanced by removing a positive charge (lysine 219) from the CuA domain.

The interaction of six different proline-rich peptides with five SH3 domains has been investigated by using spectroscopic techniques. These peptides correspond to natural sequences and have been implicated in the interaction of some SH3 domains with other proteins. We have determined the Kd values for all of the possible combinations between the peptides and the SH3 domains. Low specificity and low affinity (> 5 microM) are the most remarkable conclusions from these studies. None of the peptides tested here were found to bind with significant affinity to spectrin-SH3 or n-src-SH3. Abl-SH3 seems to be the most selective of the domains analyzed here, while Fyn-SH3 is the most promiscuous. CD and FTIR studies indicate that these peptides adopt to different extents a PPII-like structure in aqueous solution. However, analysis of the SH3 domain complexes with these peptides suggests that proline-rich peptides do not necessarily adopt an overall PPII structure over their entire length upon binding to the different SH3 domains.

Src-homology 3 (SH3) domains bind to proline-rich motifs in target proteins. We have determined high-resolution crystal structures of the complexes between the SH3 domains of Abl and Fyn tyrosine kinases, and two ten-residue proline-rich peptides derived from the SH3-binding proteins 3BP-1 and 3BP-2. The X-ray data show that the basic mode of binding of both proline-rich peptides is the same. Peptides are bound over their entire length and interact with three major sites on the SH3 molecules by both hydrogen-bonding and van der Waals contacts. Residues 4-10 of the peptide adopt the conformation of a left-handed polyproline helix type II. Binding of the proline at position 2 requires a kink at the non-proline position 3.

Cytochrome oxidase is the terminal catalyst in the mitochondrial as well as in many bacterial respiratory chains. Cytochrome c oxidase contains three redox centres, Cu A, qctochrome a, and the binuclear cytochrome a3fu B centre where oxygen is reduced to water (Fig. 1). The latter are located in subunit I, wheras Cu A resides in subunit II (for reviews, see Refs. [1-5]). Cu A has been shown to be the primary electron acceptor in cytochrome c oxidases [6]. Aerobic bacteria possess respiratory enzymes that are homologous to mitochondrial cytochrome c oxidase. Some aerobic bacteria have also homologous enzymes that use quinol instead of cytochrome c as the electron donor. Quinol oxidases are structurally and functionally related to cytochrome c oxidases, but they have lost the Cu A during evolution [7]. We have been able to reconstruct a CuA-like centre to the initially copperless subunit II (CyoA) of the E. coli cytochrome bo quinol oxidase complex. This shows that the threedimensional fold of this copper-binding domain is conserved between cytochrome c oxidases and quinol oxidases [8]. The spectroscopical characterization of Cu A has showed that it cannot be described within the classical scheme of three types of copper centre [9]. As shown in Fig. 2, the EPR spectrum of Cu A is striking similar to the spectrum of a copper centre in nitrous oxide reductase (N2OR), an enzyme involved in the reduction of nitrous oxide to dinitrogen [10,11]. N2OR is a homodimer, in which each monomer contains two copper centres (Fig. 1A). Centre A that is similar to Cu A, is

Cytochrome oxidase is a key enzyme in aerobic metabolism. All the recorded eubacterial (domain Bacteria) and archaebacterial (Archaea) sequences of subunits 1 and 2 of this protein complex have been used for a comprehensive evolutionary analysis. The phylogenetic trees reveal several processes of gene duplication. Some of these are ancient, having occurred in the common ancestor of Bacteria and Archaea, whereas others have occurred in specific lines of Bacteria. We show that eubacterial quinol oxidase was derived from cytochrome c oxidase in Gram-positive bacteria and that archaebacterial quinol oxidase has an independent origin. A considerable amount of evidence suggests that Proteobacteria (Purple bacteria) acquired quinol oxidase through a lateral gene transfer from Gram-positive bacteria. The prevalent hypothesis that aerobic metabolism arose several times in evolution after oxygenic photosynthesis, is not sustained by two aspects of the molecular data. First, cytochrome oxidase was present in the common ancestor of Archaea and Bacteria whereas oxygenic photosynthesis appeared in Bacteria. Second, an extant cytochrome oxidase in nitrogen-fixing bacteria shows that aerobic metabolism is possible in an environment with a very low level of oxygen, such as the root nodules of leguminous plants. Therefore, we propose that aerobic metabolism in organisms with cytochrome oxidase has a monophyletic and ancient origin, prior to the appearance of eubacterial oxygenic photosynthetic organisms.

The amino acid sequence obtained by translating the nucleotide sequence of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromosomal DNA by PCR, was 57% identical to part of the Escherichia coli cyoB gene, encoding subunit I of the cytochrome bo-type quinol oxidase. This fragment was mutated in vitro by insertion of a kanamycin-resistance cassette and introduced into the chromosome of A. vinelandii by homologous recombination. The mutant contained no spectrally detectable cytochrome o. However, in the stationary phase of growth, the level of the alternative oxidase (cytochrome bd) was 11-fold higher than in the wild-type strain. Respiration of the mutant was insensitive to chlorpromazine, an inhibitor thought to act specifically on cytochrome o. Cytochrome o-deficient mutants fixed nitrogen in air, clearly distinguishing the role of this oxidase from that of cytochrome bd, which is required for respiratory protection of oxygen-labile nitrogenase.

The cytochrome bc complex which is encoded by the fixNOPQ operon in Bradyrhizobiumjaponicum, is the most distant member of the haem-copper cytochrome oxidase family. We have found that its major subunit, FixN, is homologous to the NorB subunit of nitric oxide reductase in a purple bacterium. A second evolutionary link between cytochrome oxidases and denitrification enzymes is the presence of a similar binuclear copper site in cytochrome aa3 (the mitochondrial oxidase) and nitrous oxide reductase. This centre was probably acquired by a primitive FixN-type oxidase, leading to the evolution of the mitochondrial-type oxidase. These links suggest that the oxygen-reducing respiratory chain developed from the anaerobic, denitrifying respiratory system.

We previously found that the soxABCD operon encodes a quinol oxidase complex in Sulfolobus acidocaldarius and this enzyme was purified and characterized. In this study, we have used a cloning procedure based on the conservation of oxidase sequences and the polymerase chain reaction to isolate a new gene (soxM) encoding a subunit of another terminal oxidase. This terminal oxidase is a fusion between two central components of cytochrome oxidases, subunits I and III. soxM forms a transcriptional unit which is expressed under heterotrophic growth conditions. The corresponding protein was detected by direct protein sequencing in a preparation enriched with a cytochrome absorbing light at 562 nm. This preparation contains a terminal oxidase which is able to oxidize the artificial substrate N,N,N′,N′ -tetramethyl-p -phenylenediamine. This preparation also contains SoxC, a protein homologous to the mitochondrial cytochrome b, and a Rieske iron-sulphur center. We suggest that SoxM is the core component of a second terminal oxidase complex and that this complex may share a subunit (SoxC) with the SoxABCD complex.

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.

The 'pleckstrin homology' domain is an approximately 100-residue protein module that has recently been added to the domain catalogue of signalling proteins. For this review we have made an extensive database search using a profile search method, and found a number of additional proteins that may contain PH domains. The PH domain is present in many kinases, isoforms of phospholipase C, GTPases, GTPase-activating proteins and nucleotide-exchange factors, including such proteins as Vav, Dbl and Bcr, and there are two PH domains in a guanine-nucleotide releasing factor of Ras. Many PH-domain-containing proteins interact with GTP-binding proteins. We have also identified a PH domain in beta-adrenergic receptor kinase exactly in the region that has already been shown to be involved in binding to the beta and gamma subunits of a heterotrimeric G protein. This suggests that PH domains may be involved in interactions with GTP-binding proteins.

The Src-homologous SH3 domain is a small domain present in a large number of proteins that are involved in signal transduction, such as the Src protein tyrosine kinase, or in membrane-cytoskeleton interactions, but the function of SH3 is still unknown (reviewed in refs 1-3). Here we report the three-dimensional structure at 1.8 A resolution of the SH3 domain of the cytoskeletal protein spectrin expressed in Escherichia coli. The domain is a compact beta-barrel made of five antiparallel beta-strands. The amino acids that are conserved in the SH3 sequences are located close to each other on one side of the molecule. This surface is rich in aromatic and carboxylic amino acids, and is distal to the region of the molecule where the N and C termini reside and where SH3 inserts into the alpha-spectrin chain. We suggest that a protein ligand binds to this conserved surface of SH3.

Introduction The enzymes which comprise the prokaryotic and eukaryotic respiratory chains are typically membrane-bound multi-subunit complexes. In mitochondria they tend to be more complex protein conglomerates than in bacteria. In both eubacteria and archaebacteria respiratory enzymes seem to contain only the functional cores of their eukaryotic sisters. In the simplest cases they may have only a single subunit but still retain at least a part of the relevant catalytic function [ 1,2]. The archaetype of this family is the mitochondrial cytochrome c oxidiase (1.9.3.1) or cytochrome aa3, which has, depending on the species, from seven to thirteen different subunits, contains two haem A groups and two redox-active copper ions, and functions as a redox-linked proton pump [3-51. Many bacteria have a similar cytochrome aa, but this has only three or four subunits [4, 61. Different types of terminal oxidases, many of which are known to have subunits homologous to those in the mitochondrial enzyme but which contain different prosthetic groups, are frequently found in bacteria [6-81. This short review will focus on these bacterial quinol and cytochrome c oxidases.

Several loci on the Paracoccus denitrificans chromosome are involved in the synthesis of cytochrome c oxidase. So far three genetic loci have been isolated. One of them contains the structural genes of subunits II and III, as well as two regulatory genes which probably code for oxidase-specific assembly factors. In addition, two distinct genes for subunit I have been cloned, one of which is located adjacent to the cytochrome c550 gene. An alignment of six promoter regions reveals only short common sequences.

Many ATP- and GTP-binding proteins have a phosphate-binding loop (P-loop), the primary structure of which typically consists of a glycine-rich sequence followed by a conserved lysine and a serine or threonine. The three-dimensional structures of several ATP- and GTP-binding proteins containing P-loops have now been solved. In this review current knowledge of P-loops is discussed with the additional aim of illustrating the fascinating relationship between protein sequence, structure and function.

An actin-binding protein domain we call here ‘calponin-homology’ or CH is present in signalling proteins such as Vav which are involved in activation and inactivation of small G-proteins. Using profile methods, we have detected two repeats of this domain in the actin-binding region of α-actinin and related proteins. Based on this, we propose that CH domain in Vav and other signalling proteins is employed for association with filamentous actin, and that this function correlates with their control on the G-proteins Rac and Rho which are involved in the organization of cytoskeleton.

New structures of SH3 domains complexed with proline-rich peptides show that the ligands can bind in two opposite orientations; this feature has not been seen before in protein/protein recognition.

Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.

Cytochrome cbb 3 is a cytochrome c -oxidising isoenzyme that belongs to the superfamily of respiratory haem/copper oxidases. We have developed a purification method yielding large amounts of pure cbb 3 complex from the soil bacterium Pseudomonas stutzeri . This cytochrome cbb 3 complex consists of three subunits (ccoNOP) in a 1:1:1 stoichiometry and contains two b -type and three c -type haems. The protein complex behaves as a monomer with an overall molecular weight of 114.0±8.9 kDa and a s 0 20,w value of 8.9±0.3 S as determined by analytical ultracentrifugation. Crystals diffracting to 5.0 A resolution have been grown by the vapour diffusion sitting drop method to an average size of 0.1×0.1×0.3 mm. This is the first crystallisation report of a ( cbb 3 )-type oxidase.

Bacterial nitric oxide reductase (NOR), a member of the superfamily of heme-copper oxidases, catalyzes the two-electron reduction of nitric oxide to nitrous oxide. The key feature that distinguishes NOR from the typical heme-copper oxidases is the elemental composition of the dinuclear center, which contains non-heme iron (FeB) rather than copper (CuB). UV-vis electronic absorption and room-temperature magnetic circular dichroism (RT-MCD) spectroscopies showed that CO binds to Fe(II) heme b3 to yield a low-spin six-coordinate species. Photolysis of the Fe(II)-CO bond is followed by CO recombination (k(on) = 1.7 x 10(8) M(-1) x s(-1)) that is approximately 3 orders of magnitude faster than CO recombination to the active site of typical heme-copper oxidases (k(on) = 7 x 10(4) M(-1)x s(-1)). This rapid rate of CO recombination suggests an unimpeded pathway to the active site that may account for the enzyme's high affinity for substrate, essential for maintaining denitrification at low concentrations of NO. In contrast, the initial binding of CO to reduced heme b3 measured by stopped-flow spectroscopy is much slower (k(on) = 1.2 x 10(5) M(-1) x s(-1)). This suggests that an existing heme distal ligand (water/OH-) may be displaced to elicit the spin-state change observed in the RT-MCD spectrum.