Preliminary crystallographic data for cytochromes c' of Rhodopseudomonas capsulata and Rhodospirillum molischianum.

Crystallization conditions and unit cell parameters are reported for cytochromes c′ of Rhodopseudomonas capsulata and Rhodospirillum molischianum. While both proteins naturally occur as dimers having identical subunits of M_r ~ 14,000, R. capsulata was found to crystallize in the hexagonal space group P6₂ (or its enantiomorph P6₄) with one subunit per crystallographic asymmetric unit. This result suggests that the subunits of this molecule are related by exact 2-fold symmetry.     

Extended X-ray absorption fine structure studies of cytochromes c: structural aspects of oxidation-reduction

EXAFS fluorescence spectra were recorded for high-potential c-type cytochromes which range in oxidation-reduction potential from +145 to +365 mV. No average Fe-ligand bond length differences greater than 0.03 A were observed, for any cytochrome source of oxidation state. Least-squares analysis in combination with model calculations allowed limits to be set on the average Fe-N bond length (1.97-1.99 A) and the Fe-S bond length (2.29-2.32 A). A change of 0.05 A in either the average Fe-N or the Fe-S bond length is readily detectable with the range and quality of the data presented here. Two major conclusions are drawn from this study: In octahedrally coordinated iron porphyrin systems, Fe-N and Fe-S bond lengths are independent of oxidation-reduction potential, and they are also independent of oxidation state. A model for the regulation of oxidation-reduction potential in cytochrome c is proposed.     

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L₂, was investigated by small-angle X-ray scattering. In the presence of HCO ₃ ^- and Mg²⁺, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm³. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO ₃ ^- and Mg²⁺, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L₈S₈, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid     

Extended X-ray absorption fine structure studies of Zn2Fe2 hybrid hemoglobins: absence of heme bond length changes in half-ligated species

Metal hybrid hemoglobins, in which Zn(II) replaces Fe(II), have been structurally characterized by extended X-ray absorption structure (EXAFS) studies. Since Zn and Fe have very different K absorption edge energies, the structures of the ligated (Fe) and unligated (Zn) sites could be examined independently within a single molecule that mimics an intermediate ligation state. The observed EXAFS spectra and associated structural parameters are compared among the ligand free (alpha Zn)2(beta Zn)2, half-ligated (alpha FeCO)2(beta Zn)2 and (alpha Zn)2(beta FeCO)2, and fully ligated (alpha FeCO)2(beta FeCO)2 systems.     

The reaction of cytochromes c and c2 with the Rhodospirillum rubrum reaction center involves the heme crevice domain

In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 X 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 X 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.     

Extended X-ray absorption fine structure study of the coupled binuclear copper active site of tyrosinase from Neurospora crassa

Cu K-edge X-ray absorption spectra have been recorded for the enzyme tyrosinase from Neurospora crassa, in its oxy, resting (met-aquo), and inhibitor-bound (met-mimosine) forms. The K-edges proper resemble those of oxy- and met-hemocyanin, and confirm the presence of CuII. The forbidden 1s----3d transition is noticeably stronger for the 1-mimosine-bound enzyme, implying some distortion of the tetragonal Cu coordination group on inhibitor binding. The extended fine structure (EXAFS) beyond the K-edge has been analyzed. The first shell scattering is consistent with the presence of two N- and two O-ligand atoms, at 2.0 and 1.9 A, for all three forms of the enzyme; there is no evidence for heavy atom (S) scattering in the first shell. As in analogous hemocyanin derivatives, the outer shell scattering contains contributions from distant atoms of imidazole ligands, as well as from an addition scattering atom, at 3.4-3.6 A. For oxy-tyrosinase the additional scatterer is unambiguously a heavy atom (Cu), although a larger Debye-Waller factor suggests a somewhat less rigid binuclear site than in oxy-hemocyanin.     

Extended X-ray absorption fine structure studies on the iron-containing subunit of ribonucleotide reductase from Escherichia coli

Iron K-edge X-ray absorption spectra were obtained on the protein B2, the small subunit of ribonucleotide reductase from Escherichia coli. Protein B2 contains a binuclear iron center with many properties in common with the iron center of oxidized hemerythrins. The extended X-ray absorption fine structure (EXAFS) measurements on protein B2 were analyzed and compared with published data for oxyhemerythrin. In protein B2 there are, in the first coordination shell around each Fe atom, five or six oxygen or nitrogen atoms that are directly coordinated ligands. In oxyhemerythrin there are six ligands to each iron. As in oxyhemerythrin, one of the ligands in the first shell of protein B2 is at a short distance, about 1.78 A, confirming the existence of a mu-oxo bridge. The other atoms of the first shell are at an average distance of 2.04 A, which is about 0.1 A shorter than in oxyhemerythrin. In protein B2 the Fe-Fe distance is in the range 3.26-3.48 A, and the bridging angle falls between 130 and 150 degrees. On the basis of these data, there is no direct evidence for any histidine ligands in protein B2, but the noise level leaves way for the possibility of a maximum of about three histidines for each Fe pair. The X-ray absorption spectrum of a hydroxyurea-treated sample was not significantly different from that of the native protein B2, which implies that no significant alteration in the structure of the iron site occurs upon destruction of the tyrosine radical.     

5 K extended X-ray absorption fine structure and 40 K 10-s resolved extended X-ray absorption fine structure studies of photolyzed carboxymyoglobin

A previous extended X-ray absorption fine structure (EXAFS) study of photolyzed carboxymyoglobin (MbCO) [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829; Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523] has provoked much discussion on the heme structure of the photoproduct (MbCO). The EXAFS interpretation that the Fe-CO distance increases by no more than 0.05 A following photodissociation has been regarded as inconsistent with optical, infrared, and magnetic susceptibility studies [Fiamingo, F. G., & Alben, J. O. (1985) Biochemistry 24, 7964-7970; Sassaroli, M., & Rousseau, D. L. (1986) J. Biol. Chem. 261, 16292-16294]. The present experiment was performed with well-characterized dry film samples in which MbCO molecules were embedded in a poly(vinyl alcohol) matrix [Teng, T. Y., & Huang, H. W. (1986) Biochim. Biophys. Acta 874, 13-18]. The sample had a high protein concentration (12 mM) to yield adequate EXAFS signals but was very thin (40 micron) so that complete photolysis could be easily achieved by a single flash from a xenon lamp. Although the electronic state of MbCO resembles that of deoxymyoglobin (deoxy-Mb), direct comparison of EXAFS spectra indicates that structurally MbCO is much closer to MbCO than to deoxy-Mb.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extended X-ray absorption fine structure (EXAFS) studies on calcium in crystalline and amorphous solids of biological interest

Using synchroton radiation, the calcium K-edge extended x-ray absorption fine structure (EXAFS) for a number of calcium-containing solids of biological interest was measured. These included synthetic analogues of platelet dense bodies, rat tibia mineral, and synthetic bone-like calcium phosphates. The mean CaO distances in all of the solids examined were essentially the same. However, in contrast to the crystalline calcium solids studied, the dense body analogues showed sufficient structural disorder that CaP and CaCa atom pair contributions to the EXAFS spectra could not be observed. The absence of these second-shell contributions suggest that these analogues are truly amorphous rather than microcrystalline in structure. The EXAFS spectrum of the bone mineral was more similar to carbonate-apatite than to any of the other synthetic apatites studied.     

X-ray absorption fine structure study of the atomic and electronic structure of molybdenum disulfide intercalation compounds with transition metals

The local structures of ‘host’ and ‘guest’ layers of MoS₂ intercalated with M(OH)₂ (M=Mn, Co and Ni) prepared via interaction of single-layer MoS₂ dispersions and solutions of M²⁺ salts were studied by X-ray absorption spectroscopy. According to M K-edge extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) results, the electronic structure and atomic environment of the M atoms in the intercalates are similar to that of the crystalline hydroxides M(OH)₂. In the Ni intercalate, Mo K-edge EXAFS revealed a structural change of the ‘host’ MoS₂ layers similar to that reported for water dispersions of MoS₂ single layers. S K-edge XANES data indicate that the change is associated with increased electron density on the S atoms in the matrix. SO₄²⁻ and Mo″⁻ (4 < n < 6) were detected in the intercalated materials exposed to air, suggesting that transition metal intercalation may increase the susceptibility of the MoS₂ layers to oxidation.     

Determination of the nature of the heme environment in nitrosyl indoleamine 2,3-dioxygenase using Multiple-scattering analyses of X-ray absorption fine structure

Multiple-scattering analysis of X-ray absorption fine structure data on the NO adducts of indoleamine 2,3-dioxygenase (IDO) and analysis of X-ray absorption near-edge structure (XANES) have provided the first direct structural information about the iron center for this ubiquitous mammalian metalloprotein. The IDO(II)NO adduct, which is likely to play a physiological role in the immune system, differs from similar adducts such as Mb(II)NO and Lb(II)NO in that the Fe-His bond is essentially broken. At 10 K, the Fe-N(p)(av) bond length = 2.00(2) A, Fe-NO bond length = 1.75 A, and angle = 140 degrees, which are typical of five-coordinate Fe(II)NO species. The XANES is also closer to that of five-coordinate model complexes than six-coordinate species. In addition to the Fe(II)NO species, there was a minor component of the Fe(III)NO adduct because of incomplete reduction of the Fe(II) species. This was also a five-coordinate center and consists of a linear Fe(II)NO(+) moiety with the Fe-N(p)(av) bond length = 2.00(2) A, Fe-NO bond length = 1.63(3) A, and angle = 179 degrees. The results indicate that both the blocking of the heme site to O(2) binding and conformational changes induced by breaking the Fe-N(epsilon) bond may be important mechanisms by which NO inhibits IDO in vitro and in vivo.     

Extended X-ray absorption fine structure of copper in CuA-depleted, p-(hydroxymercuri)benzoate-modified, and native cytochrome c oxidase

Cytochrome c oxidase contains four redox-active metal centers: two heme irons, cytochromes a and a3, and two copper ions, CuA and CuB. Due to the paucity of spectroscopic signatures for both copper sites in cytochrome c oxidase, the ligands and structures for these sites have remained ambiguous. The specific depletion of CuA from the p-(hydroxymercuri)benzoate- (pHMB-) modified cytochrome c oxidase recently reported [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972] is herein described. Characterization of this enzyme shows that the structures of the remaining metal centers are essentially unperturbed by the CuA modification and depletion (P. M. Li, J. Gelles, and S. I. Chan, unpublished results). Copper extended X-ray absorption fine structure (EXAFS) measurements on the CuA-depleted cytochrome c oxidase reveal coordination of three (N, O) ligands and one (S, Cl) ligand at the CuB site. Comparison of EXAFS results obtained for the CuA-depleted, pHMB-modified, and "unmodified control" enzymes has allowed the deconvolution of the EXAFS in terms of the inner coordination spheres for CuA as well as CuB. On the basis of these data, it is found that the structure for the CuA site is consistent with two (N, O) ligands and two S ligands.     

Theoretical study of the ligand-CYP2B4 complexes: effect of structure on binding free energies and heme spin state

The molecular origins of temperature-dependent ligand-binding affinities and ligand-induced heme spin state conversion have been investigated using free energy analysis and DFT calculations for substrates and inhibitors of cytochrome P450 2B4 (CYP2B4), employing models of CYP2B4 based on CYP2C5(3LVdH)/CYP2C9 crystal structures, and the results compared with experiment. DFT calculations indicate that large heme-ligand interactions (ca. -15 kcal/mol) are required for inducing a high to low spin heme transition, which is correlated with large molecular electrostatic potentials (approximately -45 kcal/mol) at the ligand heteroatom. While type II ligands often contain oxygen and nitrogen heteroatoms that ligate heme iron, DFT results indicate that BP and MF heme complexes, with weak substrate-heme interactions (ca. -2 kcal/mol), and modest MEPS minima (>-35 kcal/mol) are high spin. In contrast, heme complexes of the CYP2B4 inhibitor, 4PI, the product of benzphetamine metabolism, DMBP, and water are low spin, have substantial heme-ligand interaction energies (<-15 kcal/mol) and deep MEPS minima (<-45 kcal/mol) near their heteroatoms. MMPBSA analysis of MD trajectories were made to estimate binding free energies of these ligands at the heme binding site of CYP2B4. In order to initially assess the realism of this approach, the binding free energy of 4PI inhibitor was computed and found to be a reasonable agreement with experiment: -7.7 kcal/mol [-7.2 kcal/mol (experiment)]. BP was determined to be a good substrate [-6.3 kcal/mol (with heme-ligand water), -7.3 kcal/mol (without ligand water)/-5.8 kcal/mol (experiment)], whereas the binding of MF was negligible, with only marginal binding binding free energy of -1.7 kcal/mol with 2-MF bound [-3.8 kcal/mol (experiment)], both with and without retained heme-ligand water. Analysis of the free energy components reveal that hydrophobic/nonpolar contributions account for approximately 90% of the total binding free energy of these substrates and are the source of their differential and temperature-dependent CYP2B4 binding. The results indicate the underlying origins of the experimentally observed differential binding affinities of BP and MF, and indicate the plausibility of the use of models derived from moderate sequence identity templates in conjunction with approximate free energy methods in the estimation of ligand-P450 binding affinities.     

X-ray absorption studies of myoglobin peroxide reveal functional differences between globins and heme enzymes

X-ray absorption studies of myoglobin peroxide show that although it is not identical with compound I or II of horseradish peroxidase [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611], it has some structural features in common with both. As seen in compound I, the Fe-O distance is short, but the iron-pyrrole nitrogen distance is contracted with a longer iron-histidine distance like compound II. The iron has a higher oxidation state than Fe3+, suggesting an oxyferryl ion type species. Comparison of the structures of various peroxidase and myoglobin compounds points out systematic differences that may explain the catalytic activity of the pi cation radical as well as some of the differences between globins and heme enzymes.     

Structural features and the reaction mechanism of cytochrome oxidase: iron and copper X-ray absorption fine structure.

X-ray edge absorption of copper and extended fine structure studies of both copper and iron centers have been made of cytochrome oxidase from beef heart, Paracoccus dentrificans, and HB-8 thermophilic bacteria (1-2.5 mM in heme). The desired redox state (fully oxidized, reduced CO, mixed valence formate and CO) in the x-ray beam was controlled by low temperature (-140 degrees C) and was continuously monitored by simultaneous optical spectroscopy and by electron paramagnetic resonance (EPR) monitoring every 30 min of x-ray exposure. The structure of the active site, a cytochrome a3-copper pair in fully oxidized and in mixed valence formate states where they are spin coupled, contains a sulphur bridge with three ligands 2.60 +/- 0.03 A from Fea3 and 2.18 +/- 0.03 A from Cua3. The distance between Fea3 and Cua3 is 3.75 +/- 0.05 A, making the sulphur bond angle 103 degrees reasonable for sp3 sulphur bonding. The Fea3 first shell has four typical heme nitrogens (2.01 +/- 0.03 A) with a proximal nitrogen at 2.14 +/- 0.03 A. The sixth ligand is the bridging sulphur. The Cua3 first shell is identical to oxidized stellacyanin containing two nitrogens and a bridging sulphur. Upon reduction with CO, the active site is identical to reduced stellacyanin for the Cua3 first shell and contains the sulphur that forms the bridge in fully oxidized and mixed valence formate states. The Fea3 first shell is identical to oxyhemoglobin but has CO instead of O2. The other redox centers, Fea and the other "EPR detectable" Cu are not observed in higher shells of Fea3. Fea has six equidistant nitrogens and Cua has one (or two) nitrogens and three (or two) sulphurs with typical distances; these ligands change only slight on reduction. These structures afford the basis for an oxygen reduction mechanism involving oxy- and peroxy intermediates.     

X-ray absorption and phase contrast imaging to study the interplay between plant roots and soil structure

Plant performance is, at least partly, linked to the location of roots with respect to soil structure features and the micro-environment surrounding roots. Measurements of root distributions from intact samples, using optical microscopy and field tracings have been partially successful but are imprecise and labour-intensive. Theoretically, X-ray computed micro-tomography represents an ideal solution for non-invasive imaging of plant roots and soil structure. However, before it becomes fast enough and affordable or easily accessible, there is still a need for a diagnostic tool to investigate root/soil interplay. Here, a method for detection of undisturbed plant roots and their immediate physical environment is presented. X-ray absorption and phase contrast imaging are combined to produce projection images of soil sections from which root distributions and soil structure can be analyzed. The clarity of roots on the X-ray film is sufficient to allow manual tracing on an acetate sheet fixed over the film. In its current version, the method suffers limitations mainly related to (i) the degree of subjectivity associated with manual tracing and (ii) the difficulty of separating live and dead roots. The method represents a simple and relatively inexpensive way to detect and quantify roots from intact samples and has scope for further improvements. In this paper, the main steps of the method, sampling, image acquisition and image processing are documented. The potential use of the method in an agronomic perspective is illustrated using surface and sub-surface soil samples from a controlled wheat trial. Quantitative characterization of root attributes, e.g. radius, length density, branching intensity and the complex interplay between roots and soil structure, is presented and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.