Hans C. Freeman

X-ray crystal structure analysis of plastocyanin at 2.7 Å resolution

The three-dimensional structure of plastocyanin, a ‘blue’ or ‘Type 1’ copper-protein, has been determined at a resolution of 2.7 Å. The copper atom has a highly distorted tetrahedral coordination geometry. It is coordinated by a cysteine thiol group, a methionine thioether group, and two histidine imidazole groups.

Structure of oxidized poplar plastocyanin at 1·6 Å resolution

The structure of poplar plastocyanin in the oxidized (CuII) state at pH 6.0 has been refined, using 1.6 A resolution counter data. The starting co-ordinates were obtained from the 2.7 A electron density map computed with phases derived by the multiple isomorphous replacement method. The model was refined successively by constrained real space, unrestrained reciprocal space, and restrained reciprocal space least-squares methods. The final residual R value is 0.17 for 8285 reflections (I greater than 2 sigma (I)). It is estimated that the root-mean-square standard deviation of the atomic positions is 0.1 A when averaged over all atoms, and 0.05 A for the Cu ligand atoms alone. The refined structure retains all the essential features of the 2.7 A model. The co-ordination geometry of the copper atom is confirmed as being distorted tetrahedral. The two Cu-N(His) bonds, 2.10 and 2.04 A, are within the range normally found in low molecular weight CuII complexes with Cu-N(imidazole) bonds. The Cu-S(Cys) bond, 2.13 A, is also normal, but the Cu-S(Met) bond, 2.90 A, is sufficiently long to raise important questions about its significance. The hydrogen-bonding and secondary structure can now be assigned confidently. Forty-four water molecules are included in the final model. Repetition of the refinement, using new data to 1.9 A resolution recorded from crystals at pH 4.2, has led to a residual R value of 0.16 for 6060 reflections (I greater than sigma (I)). There are few significant changes in the structure of poplar CuII-plastocyanin between pH 6.0 and pH 4.2. In particular, the geometry of the copper site is not affected. The observed changes in redox behaviour of plastocyanin at low pH are therefore unlikely to be connected with structural changes in the oxidized form of the protein. A number of features of the molecular structure appear to be directly related to the function of plastocyanin as an electron carrier in photosynthesis. Comparison between the known amino acid sequences of 67 plant plastocyanins reveals 52 conserved and 11 conservatively substituted residues in a total of 99. If three algal plastocyanin sequences are included in the comparison, there are still 26 conserved and 12 conservatively substituted residues. In many cases, the importance of these residues in determining the tertiary structure can be rationalized.

Crystal structures of metal-peptide complexes.

Publisher Summary This chapter discusses crystal structures of metal–peptide complexes. Most of the crystal-structure analyses of metal–amino acid and metal–peptide complexes have been carried out on the assumption that such complexes act as models for the metal-binding sites on proteins. Crystal-structure analyses show that the geometrical features of metal complexes of amino acids, peptides and imidazole are related in systematic ways to the chemical structure, which the complexes have in the crystalline state. Moreover, the transfer of geometrical information from crystal-structure analyses to species that exist in solution depends on the assumption that the complexes found in crystals are present also in the solutions from which the crystals grow. This chapter illustrates that, the existence of cis-trans isomers coordination isomers, optical isomers, and dimeric species in the crystalline complexes emphasizes the variety of species, which must be considered when equations are written to represent metal–peptide equilibria in solution.

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution.

BACKGROUND Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported. RESULTS The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains. The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement. All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A. CONCLUSIONS There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Crystal structure of plastocyanin from a green alga, Enteromorpha prolifera☆

The crystal structure of the Cu-containing protein plastocyanin (Mr 10,500) from the green alga Enteromorpha prolifera has been solved by molecular replacement. The structure was refined by constrained-restrained and restrained reciprocal space least-squares techniques. The refined model includes 111 solvent sites. There is evidence for alternate conformers at eight residues. The residual is 0.12 for a data set comprising 74% of all observations accessible at 1.85 A resolution. The beta-sandwich structure of the algal plastocyanin is effectively the same as that of poplar leaf (Populus nigra var. italica) plastocyanin determined at 1.6 A resolution. The sequence homology between the two proteins is 56%. Differences between the contacts in the hydrophobic core create some significant (0.5 to 1.2 A) movements of the polypeptide backbone, resulting in small differences between the orientations and separations of corresponding beta-strands. These differences are most pronounced at the end of the molecule remote from the Cu site. The largest structural differences occur in the single non-beta strand, which includes the sole turn of helix in the molecule: two of the residues in a prominent kink of the poplar plastocyanin backbone are missing from the algal plastocyanin sequence, and there is a significant change in the position of the helical segment in relation to the beta-sandwich. Several other small but significant structural differences can be correlated with intermolecular contacts in the crystals. An intramolecular carboxyl-carboxylate hydrogen bond in the algal plastocyanin may be associated with an unusually high pKa. The dimensions of the Cu site in the two plastocyanins are, within the limits of precision, identical.

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Abstract Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn2+. The EPR spectrum of Mn2+–activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5 (±0.1) Å. In the X-ray absorption spectrum of Mn2+–activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k≤12 Å–1 by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15 Å, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5 Å is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn2+–activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.

Structure of Escherichia coli aminopeptidase P in complex with the inhibitor apstatin.

Aminopeptidase P (APPro) is a metalloprotease whose active site includes a dinuclear manganese(II) cluster. The enzyme cleaves the N-terminal residue from a polypeptide when the second residue is proline. A complex of Escherichia coli APPro (EcAPPro) with an inhibitor, apstatin [N-(2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl-L-prolyl-L-prolyl-L-alaninamide], has been crystallized. Apstatin binds to the active site of EcAPPro with its N-terminal amino group coordinated to one of the two Mn(II) atoms at the metal centre. The apstatin hydroxyl group replaces a hydroxide ion which bridges the two metal atoms in the native enzyme. The first proline residue of apstatin lies in a small hydrophobic cleft. The structure of the apstatin-EcAPPro complex has been refined at 2.3 A resolution with residuals R = 0.179 and R(free) = 0.204. The structure of the complex illustrates how apstatin inhibits APPro and suggests how substrates may bind to the enzyme, but the basis of the proline-specificity remains elusive.

Preliminary crystallographic data for a basic copper-containing protein from cucumber seedlings.

A basic protein with a molecular weight of approximately 10,100 and having a single atom of Type I copper has been isolated from cucumber seedlings. The oxidized, cupric, form of the protein has been crystallized as thin olive-green plates with space group P 2 1 2 1 2 1 and cell dimensions a = 30.8 A, b = 45.6 A and c = 66.6 A. There is one protein molecule per asymmetric unit.

High resolution proton magnetic resonance studies of plastocyanin

Plastocyanin is a type I, ‘blue’ copper protein involved in photosynthetic electron transfer. It has approx. mol. wt 10 500 and contains 1 copper atom/ molecule. The copper(I1) ion may be used as an intrinsic NMR relaxation probe. The resonances of amino acids close to the copper are broadened in the oxidised protein, the effect decreasing rapidly with increasing distance from the metal. Previous NMR studies of plastocyanin from french bean [ I] , spinach and anabaena [2] have shown that certain resonances present in the spectrum of the copper (I) protein are broadened beyond detection when the protein is in the copper(I1) state. We report here the results of NMR experiments which have led to identification of several amino acid residues near the copper in french bean plastocyanin. Evidence for a high degree of conservation of residues near the copper-binding site in plastocyanins from a wide range of higher plant species is presented.

THREE NEW PLATINUM(II)-DIPEPTIDE COMPLEXES

Three new platinum(II) complexes with the dipeptide ligands l -methionylglycine, l -prolylglycine and glycyl- l -histidine have been prepared and characterized structurally. In dichloro- l -methionylglycineplatinum(II) monohydrate ( I ), coordination of l -methionylglycine to the Pt atom via the N(amino) and S(thioether) atoms leads to the formation of a six-membered chelate ring. Neither the N(peptide) nor the O(peptide) atom interacts with the metal. In chloro-dimethylsulfoxide- l -prolylglycinato-platinum(II) dihydrate ( II ), l -prolyglycine coordinates the Pt atom via the N(imino) and N(peptide) atoms to form a five-membered chelate ring. The sulfur atom of the DMSO solvent is bonded trans to the N(imino) atom of the dipeptide. In chloro-glycyl- l -histidinatoplatinum(II) monohydrate ( III ), the glycyl- l -histidine is tridentate. The Pt atom is coordinate via the N(amino), N(peptide) and N δ (imidazole) atoms so that there are adjacent five- and six-membered chelate rings. The carboxyl group of the dipeptide ligand in all three complexes I–III is protonated and does not participate in the coordination. The mechanisms of reactions that account for the formation of these complexes are discussed.