Larry C. Sieker

A crystallographic model for azurin at 3 Å resolution

Abstract The structure of the blue copper protein azurin (Mr 14,000) from Pseudomonas aeruginosa has been determined from a 3.0 A resolution electron density map computed with phases based on a uranyl derivative to 3 A resolution and a platinum derivative to 3.7 A. Interpretation of the somewhat noisy map was based on comparison of the density of the four molecules in the asymmetric unit with their averaged density. The polypeptide chain folds into an eight-strand β barrel with an additional flap containing a short helix. The copper atom is bound at one end and on the inside of the barrel, probably to a cysteine, a methionine, and two histidine residues.

Structure of a Dioxygen Reduction Enzyme from Desulfovibrio Gigas

Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 Å resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-β-lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance.

The structure of rubredoxin at 1.2 Å resolution

Structural details of the model of Clostridium pasteurianum rubredoxin are presented, based on the refined model at 1.2 A resolution. The molecule contains no extensive regions of pleated-sheet or helical structure. Regular secondary structure consists primarily of residues 3 to 7, 11 to 13 and 48 to 52 in a small region of pleated-sheet; and residues 14 to 18, 19 to 23, 29 to 33 and 45 to 49 in 310 helical corners. Interbond angles in the helical corners average as much as 10 ° greater than normally accepted values and a number of the peptide groups deviate significantly from planarity. Rubredoxin has a pronounced asymmetry in the distribution of charged groups on its surface. This would lead to highly favored molecular orientations when the protein interacts with other charged molecules. Bond lengths in the iron-sulfur complex range from 2.24 a to 2.33 A, and bond angles range from 104 ° to 114 °.

Crystallographic refinement of rubredoxin at 1·2 Å resolution

The model for rubredoxin from Clostridium pasteurianum has been refined by least-squares against a 1·2 A resolution data set. The conventional R value is 0·128 for the 10,936 reflections with I >2 σ(I) and 10 A> d >1· A. The precision of the model is much improved over the earlier refinement (Watenpaugh et al. , 1973), the mean standard deviation in the C α −C β bond lengths from the present refinment being ∼0·1 A. The Fe−S bond lengths in the FeS 4 complex range from 2·24 A to 2·33 A with an average value of 2·29 A. Standard deviations in the individual Fe−S bonds are ∼0·02 A. The B parameters, which measure the distribution of atoms about their mean positions, range from 7 A 2 to values greater than 50 A 2 . Their magnitudes are found to correlate with both inter- and intramolecular structural features, the larger B values occurring where the atoms would be expected to be less rigidly bound. The relatively large B values found for rubredoxin and characteristic of crystalline proteins generally imply relatively large amplitudes of atomic motion and pliable, readily deformable molecules. Such basic molecular properties are likely to play an important role in the mechanisms by which the molecules function. In the best determined regions of the rubredoxin molecule, hydrogen atoms are visible in the difference map.

Structures of deoxy and oxy hemerythrin at 2.0 A resolution.

The crystallographic structure analyses of deoxy and oxy hemerythrin have been carried out at 2.0 A resolution to extend the low resolution views of the physiological forms of this oxygen-binding protein. Restrained least-squares refinement has produced molecular models giving R-values of 16.8% for deoxy (41,064 reflections from 10 A to 2.0 A) and 17.3% for oxy hemerythrin (40,413 reflections from 10.0 A to 2.0 A). The protein structure in each derivative is very similar to that of myohemerythrin and the various met forms of hemerythrin. The binuclear complex in each derivative retains an oxygen atom bridging the two iron atoms, but the bond lengths found in deoxy hemerythrin support the idea that, in that form, the bridge is protonated, i.e. the bridging group is a hydroxyl. Dioxygen binds to the pentaco-ordinate iron atom in deoxy hemerythrin in the conversion to oxy hemerythrin. The interatomic distances are consistent with the proposed mechanism where the proton from the bridging group is transferred to the bound dioxygen, stabilizing it in the peroxo oxidation state by forming a hydrogen bond between the peroxy group and the bridging oxygen atom.

Structure of rubredoxin: An X-ray study to 2.5 Å resolution

Abstract The non-heme iron protein, rubredoxin (molecular weight 6300) crystallizes as rhombs which possess the symmetry of the space group R 3. The parameters of the unit cell referred to hexagonal axes are a = b = 64.5 A and c = 32.7 A with nine molecules per unit cell. The structure was first solved at 3 A resolution with data from the native protein and a single derivative (mercuri-iodide) by using the effects of anomalous scattering. The map was readily interpretable in terms of what was known about the structure. The resulting model was confirmed by a 3 A electron density map based on phases determined using a second derivative alone (uranyl derivative). Data for the native protein and the HgI 4 2− and UO 2 2+ derivatives have been extended to include all planes with spacings greater than 2.5 A. The electron density map based on these data shows that the molecule consists of an irregularly folded polypeptide chain which contains an appreciable amount of anti-parallel pleated sheet structure but no alpha helix. The iron atom is bound to four cysteine sulfur atoms to form an apparently tetrahedral complex.

Refined crystal structure of ferredoxin II from Desulfovibrio gigas at 1·7 Å☆

The crystal structure of ferredoxin II from Desulfovibrio gigas has been determined using phasing from anomalous scattering data at a resolution of 1.7 A and refined to an R-factor of 0.157. The molecule has an overall chain fold similar to that of the other bacterial ferredoxins of known structure. The molecule contains a single 3Fe-4S cluster with geometry indistinguishable from the 4Fe-4S clusters, and a disulfide bond near the site corresponding to the position of the second cluster of two-cluster ferredoxins. The cluster is bound by cysteine residues 8, 14 and 50. The side-chain of cysteine 11 extends away from the cluster, but could rotate to become the fourth cysteine ligand in the four-iron form of the molecule given a local adjustment of the polypeptide chain. This residue is modified, however, by what appears to be a methanethiol group. There are a total of eight NH . . . S bonds to the inorganic and cysteine sulfur atoms of the Fe-S cluster. There is an additional residue found that is not reported for the chemical sequence: according to the electron density a valine residue should be inserted after residue 55.

REFINEMENT OF TRICLINIC HEN EGG-WHITE LYSOZYME AT ATOMIC RESOLUTION

X-ray diffraction data have been collected at both low (120 K) and room temperature from triclinic crystals of hen egg-white lysozyme to 0.925 and 0.950 A resolution, respectively, using synchrotron radiation. Data from one crystal were sufficient for the low-temperature study, whereas three crystals were required at room temperature. Refinement was carried out using the programs PROLSQ, ARP and SHELXL to give final conventional R factors of 8.98 and 10.48% for data with F > 4sigma(F) for the low- and room-temperature structures, respectively. The estimated r.m.s. coordinate error is 0.032 A for protein atoms, 0.050 A for all atoms in the low-temperature study, and 0.038 A for protein atoms and 0.049 A for all atoms in the room-temperature case, as estimated from inversion of the blocked least-squares matrix. The low-temperature study revealed that the side chains of 24 amino acids had multiple conformations. A total of 250 waters, six nitrate ions and three acetate ions, two of which were modelled with alternate orientations were located in the electron-density maps. Three sections of the main chain were modelled in alternate conformations. The room-temperature study produced a model with multiple conformations for eight side chains and a total of 139 water molecules, six nitrate but no acetate ions. The occupancies of the water molecules were refined in both structures and this step was shown to be meaningful when assessed by use of the free R factor. A detailed description and comparison of the structures is made with reference to the previously reported structure refined at 2.0 A resolution.

Water structure in a protein crystal: Rubredoxin at 1.2 Å resolution

Abstract The model for rubredoxin based on X-ray diffraction data has been extensively refined with a 1.2 A resolution data set. Water oxygen atoms were deleted from the model if B exceeded 50 A 2 and occupancy was less than 0.3 eA −3 . The final water model consists of 127 sites with B values ranging from 15 to 6 0 A 2 and occupancies from unity down to 0.3, the most tightly bound water oxygen atoms being hydrogen bonded to two or more main-chain nitrogen or oxygen atoms. The water forms extensive hydrogen bond networks bridging the crevices on the molecular surfaces and between adjacent molecules. The minimum distances of the water sites from the protein surface are distributed about two distinct maxima, the major one at 2.5 to 3 A and a minor one at 4 to 4.5 A. Beyond 5 to 6 A from the protein surface, the discrete water merges into the aqueous continuum.

Rubredoxin in crystalline state.

Publisher Summary This chapter focuses on rubredoxin (Rd) in crystalline state, which is regarded as one of the simplest iron proteins. Rds are composed of 45 to 54 amino-acid residues with molecular weights ranging from 5000 to 6000 and contain one iron atom liganded by four cysteine residues. The iron center can be reversibly reduced at a redox potential near 0 mV. Rds are divided into three categories: (1) Rds from sulfate-reducing Desulfovibrio species, (2) Rds from a mixed assortment of bacteria, and (3) thermophilic Rds. The redox potentials of Rds isolated from sulfate-reducing bacteria (SRB) are relatively high. The amino-acid sequences of all rubredoxins show two sets of the -C- x - y -C-G- z - sequence around the iron center, where each cysteine is a ligand to the iron atom. Lys-46 is the only invariant hydrophilic residue in all the Rds. Except for the Desulfovibrio vulgaris ( D. vulgaris ) Rd structure, the crystal structures of the other Rds show that Lys-46 extends across to the neighboring chain, making an H bond to the carbonyl oxygens of residue 30 and residue 33, presumably contributing to the stability of the molecule.