Albrecht Messerschmidt

Handbook of metalloproteins

Volume 1 IRON Heme Proteins: Oxygen Storage and Oxygen Transport Proteins Heme Proteins: Cytochromes Heme Proteins: Cytochrome Peroxidases Heme Proteins: Cytochrome P-450 Heme Proteins: Oxidoreductases Non-Heme Proteins: Iron-Sulfur Clusters Non-Heme Proteins: Mononuclear Iron Proteins Volume 2 IRON continued Non-Heme Proteins: Dinuclear Iron Proteins Non-Heme Proteins: Iron Storage Non-Heme Proteins: Iron Transport NICKEL MANGANESE COBALT MOLYBDENUM/TUNGSTEN COPPER Cupredoxins (Type-1 Copper Proteins) Type-2 Copper Enzymes Binuclear Copper: Type-3 Copper Enzymes Binuclear Copper: CuA Copper Multicopper Enzymes Copper Storage and Transport VANADIUM.

Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5.5 and pH 9.0. A pH-induced conformational transition involves a peptide bond flip.

The X-ray crystal structure of recombinant wild-type azurin from Pseudomonas aeruginosa was determined by difference Fourier techniques using phases derived from the structure of the mutant His35Leu. Two data sets were collected from a single crystal of oxidized azurin soaked in mother liquor buffered at pH 5.5 and pH 9.0, respectively. Both data sets extend to 1.93 A resolution. The two pH forms were refined independently to crystallographic R-factors of 17.6% (pH 5.5) and 17.5% (pH 9.0). The conformational transition previously attributed to the protonation/deprotonation of residue His35 (pKa(red) = 7.3, pKa(ox) = 6.2), which lies in a crevice of the protein close to the copper binding site, involves a concomitant Pro36-Gly37 main-chain peptide bond flip. At the lower pH, the protonated imidazole N delta 1 of His35 forms a strong hydrogen bond with the carbonyl oxygen from Pro36, while at alkaline pH the deprotonated N delta 1 acts as an acceptor of a weak hydrogen bond from HN Gly37. The structure of the remainder of the azurin molecule, including the copper binding site, is not significantly affected by this transition.

Refined crystal structure of ascorbate oxidase at 1.9 A resolution.

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)

X-ray crystal structure of the blue oxidase ascorbate oxidase from Zucchini: Analysis of the polypeptide fold and a model of the copper sites and ligands

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.

Structure of cytochrome c nitrite reductase

The enzyme cytochrome c nitrite reductase catalyses the six-electron reduction of nitrite to ammonia as one of the key stepsin the biological nitrogen cycle, where it participates inthe anaerobic energy metabolism of dissimilatory nitrate ammonification. Here we report on the crystal structure of this enzyme from the microorganism Sulfurospirillum deleyianum, which we solved by multiwavelength anomalous dispersion methods. We propose a reaction scheme for the transformation of nitrite based on structural and spectroscopic information. Cytochrome c nitrite reductase is a functional dimer, with 10 close-packed haem groups of type c and an unusual lysine-coordinated high-spin haem at the active site. By comparing the haem arrangement of this nitrite reductase with that of other multihaem cytochromes, we have been able to identify a family of proteins in which the orientation of haem groups is conserved whereas structure and function are not.

Implications for the catalytic mechanism of the vanadium-containing enzyme chloroperoxidase from the fungus Curvularia inaequalis by X-ray structures of the native and peroxide form.

Implications for the catalytic mechanism of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis have been obtained from the crystal structures of the native and peroxide forms of the enzyme. The X-ray structures have been solved by difference Fourier techniques using the atomic model of the azide chloroperoxidase complex. The 2.03 A crystal structure (R = 19.7%) of the native enzyme reveals the geometry of the intact catalytic vanadium center. The vanadium is coordinated by four non-protein oxygen atoms and one nitrogen (NE2) atom from histidine 496 in a trigonal bipyramidal fashion. Three oxygens are in the equatorial plane and the fourth oxygen and the nitrogen are at the apexes of the bipyramid. In the 2.24 A crystal structure (R = 17.7%) of the peroxide derivate the peroxide is bound to the vanadium in an eta2-fashion after the release of the apical oxygen ligand. The vanadium is coordinated also by 4 non-protein oxygen atoms and one nitrogen (NE2) from histidine 496. The coordination geometry around the vanadium is that of a distorted tetragonal pyramid with the two peroxide oxygens, one oxygen and the nitrogen in the basal plane and one oxygen in the apical position. A mechanism for the catalytic cycle has been proposed based on these X-ray structures and kinetic data.

Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane‐embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenyl‐pyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD‐binding domain of the p‐hydroxybenzoate‐hydrolase fold and a substrate‐binding domain that enclose a narrow active site cavity beneath the FAD and an α‐helical membrane‐binding domain. The active site architecture suggests a specific substrate‐binding mode compatible with the unusual six‐electron oxidation. The membrane‐binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO.

Cytochrome c nitrite reductase from Wolinella succinogenes. Structure at 1.6 A resolution, inhibitor binding, and heme-packing motifs.

Cytochrome c nitrite reductase catalyzes the 6-electron reduction of nitrite to ammonia. This second part of the respiratory pathway of nitrate ammonification is a key step in the biological nitrogen cycle. The x-ray structure of the enzyme from the ε-proteobacterium Wolinella succinogenes has been solved to a resolution of 1.6 Å. It is a pentahemec-type cytochrome whose heme groups are packed in characteristic motifs that also occur in other multiheme cytochromes. Structures of W. succinogenes nitrite reductase have been obtained with water bound to the active site heme iron as well as complexes with two inhibitors, sulfate and azide, whose binding modes and inhibitory functions differ significantly. Cytochrome cnitrite reductase is part of a highly optimized respiratory system found in a wide range of Gram-negative bacteria. It reduces both anionic and neutral substrates at the distal side of a lysine-coordinated high-spin heme group, which is accessible through two different channels, allowing for a guided flow of reaction educt and product. Based on sequence comparison and secondary structure prediction, we have demonstrated that cytochromec nitrite reductases constitute a protein family of high structural similarity.

Crystallization, crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae

The bilin binding protein of the butterfly Pieris brassicae has been prepared, crystallized and its crystal structure determined at high resolution using film and FAST area detector intensity data. The crystallographic asymmetric unit contains a tetramer of identical subunits with a molecular weight of about 90,000. The crystal structure was determined by isomorphous replacement. Use was made of the molecular symmetry to improve phases. A molecular interpretation of the electron density distribution and partial tracing of the polypeptide chain was possible without amino acid sequence information, as the fold is very similar to retinol binding protein. It is characterized by a beta-barrel formed by two orthogonal beta-sheets and an alpha-helix. The bilin pigment seems to be bound within the beta-barrel analogously to retinol in retinol binding protein. The tetramer in the crystal has C2 symmetry and is a dimer of dimers of quasi-equivalent subunits.

X-ray crystal structure of the two site-specific mutants His35Gln and His35Leu of azurin from Pseudomonas aeruginosa.

The three-dimensional structures of two site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa have been solved by a combination of isomorphous replacement and Patterson search techniques, and refined by energy-restrained least-squares methods. The mutations introduced by recombinant DNA techniques involve residue His35, which was exchanged for glutamine and leucine, to probe for its suggested role in electron transfer. The two mutants, His35Gln (H35Q) and His35Leu (H35L), crystallize non-isomorphously in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 109.74 A, b = 99.15 A, c = 47.82 A for H35Q, a = 57.82 A, b = 81.06 A, c = 110.03 A for H35L. In each crystal form, there are four molecules in the asymmetric unit. They are arranged as a dimer of dimers in the H35Q case and are distorted from ideal C2 symmetry in H35L. The final crystallographic R-value is 16.3% for 20.747 reflections to a resolution of 2.1 A for H35Q and 17.0% for 32,548 reflections to 1.9 A for H35L. The crystal structures reported here represent the first crystallographically refined structures for azurin from P. aeruginosa. The structure is very similar to that of azurin from Alcaligenes denitrificans. The copper atom is located about 7 A below a hydrophobic surface region and is ligated by five donor groups in a distorted trigonal bipyramidal fashion. The implications for electron transfer properties of the protein are discussed in terms of the mutation site and the packing of the molecules within the tetramer.