Biomedical subjects

H C Freeman

Publications and source records attributed to H C Freeman.

At least 19 recordsLinked to original sources

PubMed1999

The structure of plastocyanin from the cyanobacterium Phormidium laminosum.

The crystal structure of the 'blue' copper protein plastocyanin from the cyanobacterium Phormidium laminosum has been solved and refined using 2.8 A X--ray data. P. laminosum plastocyanin crystallizes in space group P43212 with unit-cell dimensions a = 86.57, c = 91.47 A and with three protein molecules per asymmetric unit. The final residual R is 19.9%. The structure was solved using molecular replacement with a search model based on the crystal structure of a close homologue, Anabaena variabilis plastocyanin (66% sequence identity). The molecule of P. laminosum plastocyanin has 105 amino-acid residues. The single Cu atom is coordinated by the same residues - two histidines, a cysteine and a methionine - as in other plastocyanins. In the crystal structure, the three molecules of the asymmetric unit are related by a non-crystallographic threefold axis. A Zn atom lies between each pair of neighbouring molecules in this ensemble, being coordinated by a surface histidine residue of one molecule and by two aspartates of the other.

Amino Acid Sequence↗

PubMed1998

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli.

The structure of the proline-specific aminopeptidase (EC 3.4.11.9) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-A resolution, for a dipeptide-inhibited complex at 2.3-A resolution, and for a low-pH inactive form at 2.7-A resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.

Aminopeptidases↗

PubMed1997

Crystal structures of the copper-containing amine oxidase from Arthrobacter globiformis in the holo and apo forms: implications for the biogenesis of topaquinone.

The crystal structures of the copper enzyme phenylethylamine oxidase from the Gram-positive bacterium Arthrobacter globiformis (AGAO) have been determined and refined for three forms of the enzyme: the holoenzyme in its active form (at 2.2 A resolution), the holoenzyme in an inactive form (at 2.8 A resolution), and the apoenzyme (at 2.2 A resolution). The holoenzyme has a topaquinone (TPQ) cofactor formed from the apoenzyme by the post-translational modification of a tyrosine residue in the presence of Cu2+. Significant differences between the three forms of AGAO are limited to the active site. The polypeptide fold is closely similar to those of the amine oxidases from Escherichia coli [Parsons, M. R., et al. (1995) Structure 3, 1171-1184] and pea seedlings [Kumar, V., et al. (1996) Structure 4, 943-955]. In the active form of holo-AGAO, the active-site Cu atom is coordinated by three His residues and two water molecules in an approximately square-pyramidal arrangement. In the inactive form, the Cu atom is coordinated by the same three His residues and by the phenolic oxygen of the TPQ, the geometry being quasi-trigonal-pyramidal. There is evidence of disorder in the crystals of both forms of holo-AGAO. As a result, only the position of the aromatic group of the TPQ cofactor, but not its orientation about the Cbeta-Cgamma bond, is determined unequivocally. In apo-AGAO, electron density consistent with an unmodified Tyr occurs at a position close to that of the TPQ in the inactive holo-AGAO. This observation has implications for the biogenesis of TPQ. Two features which have not been described previously in amine oxidase structures are a channel from the molecular surface to the active site and a solvent-filled cavity at the major interface between the two subunits of the dimer.

Amine Oxidase (Copper-Containing)↗

PubMed1996

The structure of a phytocyanin, the basic blue protein from cucumber, refined at 1.8 A resolution.

The crystal structure of the cucumber basic protein (CBP), a type 1 or blue copper protein, has been refined at 1.8 A resolution. The molecule resembles other blue copper proteins in having a Greek key beta-barrel structure, except that the barrel is open on one side and is better described as a "beta-sandwich" or "beta-taco". The Cu atom has the normal blue copper NNSS' co-ordination with bond lengths Cu-N(His39) = 1.93 A, Cu-S(Cys79) = 2.16 A, Cu-N(His84) = 1.95 A, Cu-S(Met89) = 2.61 A. The Cu-S(Met) bond is the shortest so far observed in a blue copper protein. A disulphide link, (Cys52)-S-S-(Cys85), appears to play an important role in stabilising the molecular structure. It is suggested that the polypeptide fold is typical of a sub-family of blue copper proteins (phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3, with which CBP has a high degree of sequence identify. The proteins currently identifiable as phytocyanins are CBP, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana. In all except CBP and the pea-pod protein, the axial methionine ligand normally found at blue copper sites is replaced by glutamine. The structure of CBP was originally solved by the multiple wavelength anomalous scattering method, using data recorded at four wavelengths. All these data were included in the restrained least squares refinement. The final model comprises 96 amino acid residues, 122 solvent molecules and a copper atom. Several residues are modelled as having more than one conformation. The residual R is 0.141 for 41,910 observations (including Bijvoet-related observations) of 8.142 unique reflections in the resolution range 7 to 1.8 A.

Allergens↗

PubMed1996

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.

Amine Oxidase (Copper-Containing)↗

PubMed1993

Crystallization and preliminary crystallographic characterization of the copper-containing amine oxidase from pea seedlings.

The copper-containing amine oxidase from pea seedlings has been crystallized using lithium sulfate as precipitant at pH 5.2. The unit cell is orthorhombic, space group P2(1)2(1)2(1), with dimensions a = 89.3 A, b = 113.4 A, c = 199.0 A. The mass of the asymmetric unit is 131(+/- 13) kDa, consistent with independent evidence that the molecule has two approximately 66 kDa subunits. The crystals diffract to 2.5 A in a synchrotron X-ray beam.

Amine Oxidase (Copper-Containing)↗

PubMed1992

Accuracy and precision in protein structure analysis: restrained least-squares refinement of the structure of poplar plastocyanin at 1.33 A resolution.

The structure of the electron-transfer protein, plastocyanin (99 amino acids, one Cu atom, 10,500 Da) from poplar leaves, has been refined at 1.33 A resolution to a residual R = 0.15. The space group is orthorhombic, P2(1)2(1)2(1), a = 29.60 (1), b = 46.86 (3), c = 57.60 (3) A. The 14,303 reflections used in the refinement were obtained from a data set recorded on a four-circle diffractometer with radiation from a sealed fine-focus tube, combined with a data set measured on oscillation films exposed at the DESY synchrotron. The final model comprises 1442 (738 non-H) protein atoms, one Cu atom and 110 solvent molecules. Nine residues are described as disordered. The root-mean-square deviation from ideal bond lengths is 0.016 A and the root-mean-square difference between the positions of the C alpha atoms in this refined model and in the structure previously refined at 1.6 A resolution is 0.11 A. The effects of manual model adjustment, resolution, choice of standard values for geometrical parameters, inclusion of H atoms and inclusion of anomalous-scattering corrections on the copper-site geometry have been explored. The final values of the Cu-ligand bond lengths are: Cu--N(His37) 1.91, Cu--S(Cys84) 2.07, Cu--N(His87) 2.06, Cu--S(Met92) 2.82 A.

Amino Acid Sequence↗

PubMed1991

Three-dimensional model for stellacyanin, a "blue" copper-protein.

A three-dimensional model of the "blue" copper-glycoprotein stellacyanin from Rhus vernicifera has been derived by computer graphics, energy minimization and molecular dynamics techniques. The initial atomic co-ordinates were obtained by making substitutions and insertions in the known structure of another blue copper-protein, cucumber basic protein (CBP), which is 46% homologous with stellacyanin and has similar spectroscopic properties. An important difference between CBP and stellacyanin is that the latter lacks methionine, a residue that forms an exceptionally long bond to the copper atom in all blue copper-proteins of known structure. In the aligned amino acid sequences, stellacyanin has glutamine 97 at the position that corresponds to the copper-binding methionine 89 in CBP. The hypothesis that the copper atom in stellacyanin is co-ordinated by the side-chain functional groups of histidine 46, cysteine 87, histidine 92 and glutamine 97 leads to a model that enables the spectroscopic properties, redox potential and electron-transfer kinetics of the protein to be rationalized. The present model for stellacyanin is more plausible than an antecedent model derived from the structure of plastocyanin. This demonstrates that the output from molecular modeling calculations is strongly dependent on the input, and that sequence homology with the target molecule is an important criterion for the selection of a starting model.

Amino Acid Sequence↗

PubMed1991

Resonance Raman spectra of plastocyanin and pseudoazurin: evidence for conserved cysteine ligand conformations in cupredoxins (blue copper proteins).

New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.

Azurin↗

PubMed1991

Electrostatic orientation of the electron-transfer complex between plastocyanin and cytochrome c.

To understand the specificity and efficiency of protein-protein interactions promoting electron transfer, we evaluated the role of electrostatic forces in precollision orientation by the development of two new methods, computer graphics alignment of protein electrostatic fields and a systematic orientational search of intermolecular electrostatic energies for two proteins at present separation distances. We applied these methods to the plastocyanin/cytochrome c interaction, which is faster than random collision, but too slow for study by molecular dynamics techniques. Significant electrostatic potentials were concentrated on one-fourth (969 A2) of the plastocyanin surface, with the greatest negative potential centered on the Tyr-83 hydroxyl within the acidic patch, and on one-eighth (632 A2) of the cytochrome c surface, with the greatest positive potential centered near the exposed heme edge. Coherent electrostatic fields occurred only over these regions, suggesting that local, rather than global, charge complementarity controls productive recognition. The three energetically favored families of pre-collision orientations all directed the positive region surrounding the heme edge of cytochrome c toward the acidic patch of plastocyanin but differed in heme plane orientation. Analysis of electrostatic fields, electrostatic energies of precollision orientations with 12 and 6 A separation distances, and surface topographies suggested that the favored orientations should converge to productive complexes promoting a single electron-transfer pathway from the cytochrome c heme edge to Tyr-83 of plastocyanin. Direct interactions of the exposed Cu ligand in plastocyanin with the cytochrome c heme edge are not unfavorable sterically or electrostatically but should occur no faster than randomly, indicating that this is not the primary pathway for electron transfer.

Amino Acid Sequence↗

PubMed1990

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.

Amino Acid Sequence↗

PubMed1988

Phase determination by multiple-wavelength x-ray diffraction: crystal structure of a basic "blue" copper protein from cucumbers.

A novel x-ray diffraction technique, multiple-wavelength anomalous dispersion (MAD) phasing, has been applied to the de novo determination of an unknown protein structure, that of the "blue" copper protein isolated from cucumber seedlings. This method makes use of crystallographic phases determined from measurements made at several wavelengths and has recently been made technically feasible through the use of intense, polychromatic synchrotron radiation together with accurate data collection from multiwire electronic area detectors. In contrast with all of the conventional methods of solving protein structures, which require either multiple isomorphous derivatives or coordinates of a similar structure for molecular replacement, this technique allows direct solution of the classical "phase problem" in x-ray crystallography. MAD phase assignment should be particularly useful for determining structures of small to medium-sized metalloproteins for which isomorphous derivatives are difficult or impossible to make. The structure of this particular protein provides new insights into the spectroscopic and redox properties of blue copper proteins, an important class of metalloproteins widely distributed in nature.

Amino Acid Sequence↗

PubMed1988

In vitro biosynthesis of 17 alpha,20 alpha,20 beta-dihydroxy-4-pegnen-3-one by the ovaries, testes, and head kidneys of the Atlantic salmon Salmo salar.

Ovaries, testes, and head kidneys of sexually mature Atlantic salmon, Salmo salar, biosynthesized 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHP) from equimolar amounts of [3H]pregnenolone plus [4-14C]progesterone in vitro. The 3H:14C isotope ratios of steroid metabolites indicated that the biosynthetic pathways to 17 alpha,20 beta-diOHP in the testes differed from those observed in the ovaries and head kidneys. [4-14C]Progesterone appeared to be the principal precursor of 17 alpha,20 beta-diOHP in the testes, whereas both precursors were efficiently biotransformed to 17 alpha,20 beta-diOPH in the ovaries and head kidneys. 17 alpha-Hydroxy-4-pregnen-3-one (17 alpha-OHP) was the immediate precursor to 17 alpha,20 beta-diOHP in all tissues. However, appreciable amounts of 17 alpha,20 beta-diOHP accumulated in vitro in the testes only in the presence of exogenous [14C]progesterone. Incubation of the testes, ovaries, and head kidneys with [14C]pregnenolone resulted in high yields of 17 alpha,20 beta-diOHP in the ovaries and head kidneys but no detectable amounts of the steroid in the testes. The results confirm that progesterone is the favored precursor to 17 alpha,20 beta-diOHP in the testes. The results also suggest that the head kidneys may be an excellent cellular source of 17 alpha,20 beta-diOHP in both male and female. Atlantic salmon and may play an important role in the sexual maturation process in this fish. It is suggested that biosynthetic control mechanism affecting 17 alpha,20 beta-diOHP synthesis and/or spermiation and ovulation may differ in male and female Atlantic salmon.

Animals↗

PubMed1987

A simple, rapid, and precise direct radioimmunoassay method for 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one in salmon plasma.

This report describes a radioimmunoassay method for 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHP) in the blood plasma of Atlantic salmon (Salmo salar). 3H-labeled 17 alpha,20 beta-diOHP was synthesized from 17 alpha-[3H]hydroxy-4-pregnen-3-one (17 alpha-OHP) by enzymatic reduction of the C-20 oxo group and the simultaneous oxidation of coenzyme-reduced nicotinamide adenine dinucleotide. Antiserum to 17 alpha,20 beta-diOHP-3-CMO-BSA exhibited high specificity: all steroids tested against the antiserum gave less than 1% cross-reactivity at 50% displacement. Crude dichloromethane extracts of small volumes (50 microliters or less) of plasma can be assayed directly without prior purification. The working range is 5 to 300 pg. Interassay and intraassay coefficients of variation were less than 10%. Various parallelism tests showed high accuracy and sensitivity. The assay is simple and rapid and at least 75 samples can be easily assayed in duplicate by two analysts in 1 day.

Animals↗

PubMed1986

Crystal structure analyses of reduced (CuI) poplar plastocyanin at six pH values.

The structure of poplar plastocyanin in the reduced (CuI) state has been determined and refined, using counter data recorded from crystals at pH 3.8, 4.4, 5.1, 5.9, 7.0 and 7.8 (resolution 1.9 A, 1.9 A, 2.05 A, 1.7 A, 1.8 A and 2.15 A; the final residual R value was 0.15, 0.15, 0.16, 0.17, 0.16 and 0.15, respectively). The molecular and crystal structure of the protein is substantially the same in the reduced state as in the oxidized state. The refinements of the structures of the six forms of the reduced protein could therefore be commenced with a model derived from the known structure of CuII-plastocyanin. The refinements were made by reciprocal space least-squares calculations interspersed with inspections of electron-density difference maps. Precautions were taken to minimize any bias of the results of the refinements in the direction of the starting model. The most significant differences among the structures of the reduced protein at the six pH values, or between them and the structure of the oxidized protein, are concentrated at the Cu site. In the reduced protein at high pH (pH 7.8), the CuI atom is co-ordinated by the N delta(imidazole) atoms of His37 and His87, the S gamma(thiolate) atom of Cys84, and the S delta(thioether) atom of Met92, just as in CuII-plastocyanin. The distorted tetrahedral geometry and the unusually long Cu-S(Met92) bond are retained. The only effects of the change in oxidation state are a lengthening of the two Cu-N(His) bonds by about 0.1 A, and small changes in two bond angles involving the Cu-S(Cys) bond. The high-pH form of reduced plastocyanin accordingly meets all the requirements for efficient electron transfer. As the pH is lowered, the Cu atom and the four Cu-binding protein side-chains appear to undergo small but concerted movements in relation to the rest of the molecule. At low pH (pH 3.8), the CuI atom is trigonally co-ordinated by N delta(His37), S gamma(Cys84) and S delta(Met92). The fourth Cu-ligand bond is broken, the Cu atom making only a van der Waals' contact with the imidazole ring of His87. The trigonal geometry of the Cu atom strongly favours CuI, so that this form of the protein should be redox-inactive. This is known to be the case.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1986

Complete amino acid sequence of plastocyanin from a green alga, Enteromorpha prolifera.

The complete amino acid sequence of the plastocyanin from the green alga Enteromorpha prolifera has been determined by Edman degradation of the intact molecule and fragments produced by enzymatic cleavage of the polypeptide chain with chymotrypsin, Staphylococcus aureus protease, proline-specific endopeptidase, Lys-C endopeptidase and trypsin. The molecule consists of 98 amino acid residues with a calculated relative molecular mass of 10103. The amino acid sequence of E. prolifera plastocyanin shows a high degree of homology with those plastocyanins from other algae and higher plants. In particular, the four residues which are copper ligands in other plastocyanins and in the bacterial electron transport protein azurin (two histidines, one cysteine and one methionine) are conserved. Five out of the six acidic amino acid side-chains which create an 'acidic patch' on the surface of plastocyanin from Populus nigra var. italica [Colman, P. M. et al. (1978) Nature (Lond.) 272, 319-324] are conserved in the amino acid sequence of E. prolifera plastocyanin.

Amino Acid Sequence↗

PubMed1986

The crystal structure of mercury-substituted poplar plastocyanin at 1.9-A resolution.

The crystal structure of Hg(II)-plastocyanin has been determined and refined at a resolution of 1.9 A. The crystals were prepared by soaking crystals of Cu(II)-plastocyanin from poplar leaves (Populus nigra var. italica) in a solution of a mercuric salt. Replacement of the Cu(II) atom in plastocyanin by Hg(II) causes only minor changes in the geometry of the metal site, and there are few significant changes elsewhere in the molecule. It is concluded that, as in the case of the native protein, the geometry of the metal site is determined by the polypeptide. The weak metal-S(methionine) bond found in Cu(II)-plastocyanin remains weak in Hg(II)-plastocyanin. The "flip" of a proline side chain close to the metal site from a C gamma-exo conformation in Cu(II)-plastocyanin to a C gamma-endo conformation in Hg(II)-plastocyanin suggests that this region of the molecule is particularly flexible. Crystallographic evidence for the close similarity of the Hg(II)- and Cu(II)-plastocyanin structures was originally obtained from electron density difference maps at 2.5-A resolution. The refinement of the structure was begun with a set of atomic coordinates taken from the structure of Cu(II)-plastocyanin. A Hg(II) atom was substituted for the Cu(II) atom, and the side chains of 6 residues in the vicinity of the metal site were omitted. Three series of stereochemically restrained least-squares refinement calculations were interspersed with two stages of model adjustment followed by phase extension. Fifty-nine water molecules were located. The final structure has a crystallographic residual R = 0.16.

Copper↗

PubMed1984

The crystal structure of poplar apoplastocyanin at 1.8-A resolution. The geometry of the copper-binding site is created by the polypeptide.

The three-dimensional structure of apoplastocyanin from poplar leaves (Populus nigra var. italica) has been determined by x-ray diffraction at 1.8-A resolution. The structure closely resembles that of the holoprotein. In particular, the positions of the copper-binding residues in the apo- and holoproteins differ by only 0.1-0.3 A. This indicates that the irregular geometry of the "type 1" copper site is imposed upon the metal atom by the polypeptide moiety. A 180 degrees rotation of one solvent-exposed histidine imidazole ring about C beta-C gamma appears to facilitate access to the copper site. The close structural similarity between apo-, Cu-(II)-, and Cu(I)-plastocyanin was initially demonstrated by means of electron density difference maps. Two series of restrained least squares refinement calculations for apoplastocyanin, originating from different sets of atomic positional parameters, were carried out in parallel. Both refinements converged to the same model which, when fully refined, had a residual R = 0.16. Forty-two water molecules were located during the refinement.

Apoproteins↗