Models for metal-sulfur coordination in copper proteins.

The oxidation state of copper in several model compounds containing copper coordinated to sulfur, some of which are suggested model compounds for naturally occurring Cu-S chromophores, was determined employing x-ray photoelectron spectrometry. Copper was found to be present in a 3d10 state, which is characteristic for Cu(I). An initial photoreduction is excluded, as Cu(ethylenediamine)2 (SCN)2, in which a Cu-S bond is also present, was found to contain Cu(II) (3d9). As was the case with native parsley plastocyanin, the model compounds reacted effectively with superoxide anion radicals. We suggest that the electrons of the metal-sulfur chromophore in blue copper proteins are delocalized and that an equilibrium: RS--Cu(II) in equilibrium or formed from RS.Cu(I) exists.     

Elementary reactions,structure-function relationships,and the potential relevance of low molecular weight metal-sulfur ligand complexes to biological N2 fixation

This article tries to rationalize the shortcomings of various model compounds and discusses requirements that a low-molecular compound must fulfill in order to become a potentially competitive catalyst for nitrogenases. For fundamental reasons, such a synthetic catalyst cannot be a precise structural duplicate of the active centers of nitrogenase. Results obtained with iron-sulfur carbonyl and diazene complexes further indicate that (1) the coupling and chronology of proton and electron transfer steps, (2) invariance of iron-sulfur distances within a wide range of electron density changes at the iron centers, and (3) Brönsted basic thiolate donors favoring the protonation of metal-sulfur cores and the formation of N–H···S bridges may be essential in order to reduce N₂ via N₂H₂ and N₂H₄ to NH₃ under mild conditions.     

Metal-mediated reduction of cytochrome c by thioglycolic acid

The reduction of cytochrome c by thioglycolic acid was found to be extremely sensitive to metal catalysis. The rate of the uncatalyzed reaction was negligible in comparison with rates obtained from reactions supplemented with catalytic amounts of copper or iron. Both the catalyzed and uncatalyzed reactions were independent of pH (near neutrality) but when o-phenanthroline was included in the reaction mixture, a pH dependence was induced. This pH dependence is the result of an interference of oxygen with the metal complexes. A comparison of the rate constants at zero ionic strenght, which were obtained from the application of the Debye-Hückel theory for the ionic strength dependence, demonstrated that copper complexes are superior catalysts as compared with iron complexes. Our results suggest that in the copper-mediated reaction, the catalyst is a cupric thioglycolate complex with a net charge of ?2. The addition of o-phenanthroline to the reaction mixture results in a tenfold decrease in the catalytic activity and in a change in the net charge of the catalyst to ?1. At pH 8 the iron-mediated reduction is catalyzed by a ferric thioglycolate complex, whereas at pH 7 a ferrothioglycolate complex provides the catalytic activity. Both complexes have a net negative charge of ?2. At both pH's the catalytic activity is completely abolished by the addition of o-phenanthroline. The results demonstrate the effectiveness by which metal-sulfur complexes can facilitate one-electron transfer reactions and could there-fore serve as a model in the study of various biological oxidations.     

The linear electric field effect in stellacyanin, azurin and in some simple model compounds.

All mononuclear Cu(II) sites in frozen solution are non-centrosymmetric and, unless physically constrained, will have a tetrahedral distortion away from the usual square planar structure often presented for Cu(II) complexes. Blue copper sites such as are found in azurin and stellacyanin have a greater distortion towards a tetrahedral geometry than do simple Cu(II) complexes. The distortion is comparable to that which is observed for Cu(II)-o-phenanthroline dichloride, a known tetrahedral complex. Blue copper sites possess an axis of asymmetry directed away from g parallel which could arise from a metal-sulfur interaction.     

Different reaction behaviour of molybdenum and tungsten - Reactions of the dichloro dioxo dimethyl-bispyridine complexes with thiophenolate

The reaction of the metal complexes MO₂Cl₂(mebipy) (M = Mo, W) with two equivalents thiophenol by the exact same procedure leads to two different products for molybdenum [Mo₂O₄(SPh)₂(mebipy)₂] and tungsten [WO₂(SPh)₂(mebipy)]. To understand why this is the case the redox potentials of the starting materials were measured showing that the redox potential for thiophenol is lower than the redox potentials (M^V ↔ M^VI) for both of the metal precursors. A reduction of the metal and oxidation of the sulfur should be possible for both reactions but occurs only for the molybdenum compound. Theoretical calculations show that different metal-sulfur bond strengths are as well and equally responsible for the differing reaction behaviour as are the redox potentials.     

用距离几何算法和同源建模法对水稻类金属硫蛋白（rgMT）的三维结构建模

水稻类金属硫蛋白(rgMT)的两端是高度保守的半胱氨酸富含区的结构域(CR区),中间是不含半胱氨酸的间隔区,呈典型的三段式结构。本研究分别采用距离几何算法和同源建模相结合的方法对水稻类金属硫蛋白进行三级结构建模。在排列出CR区的所有可能的半胱氨酸-金属硫络合的组合方式,并对每一种组合方式给出一定的限制条件后各生成20个随机构象。根据生成的随机构象足否能形成金属硫络合结构,从900个随机构象中最终选出6个构象(N端4种,C端2种组合)作为可能的结构模型。另一方面,采用GOR方法对间隔区进行了二级结构预测,随后用同源建模法对其建模。将上述建成的三部分模型连接起来后形成rgMT的整体三维构象。结果表明rgMT能像哺乳动物MT蛋白一样,可形成两个独立的、在结构和能量上均没有障碍的金属-硫络合结构。介于所有植物类金属硫蛋白都具有典型的三段式结构,其中的一部分还具有与rgMT相同的半胱氨酸排列方式,所以rgMT三维结构模型的建立对于其他植物类金属硫蛋白的结构研究具有重要的参考价值。     

X-ray absorption spectroscopy of Pyrococcus furiosus rubredoxin

X-ray absorption spectroscopy has been used to probe the frozen solution structure of the metal site in Pyrococcus furiosus rubredoxin in the native, iron-containing protein and in zinc- and mercury-substituted proteins. For all samples studied, the spectra have been interpreted in terms of a single shell of coordinated sulfur, with approximately tetrahedral coordination. For the native protein we obtain Fe-S bond-lengths of 2.29 and 2.33?Å for oxidized and reduced proteins, respectively. These values are in excellent agreement with those previously obtained from X-ray crystallography. The metal-substituted rubredoxins possess metal-sulfur bond lengths of 2.34 and 2.54?Å for the zinc- and mercury-substituted proteins, respectively.     

用距离几何算法和同源建模法对水稻类金属硫蛋白(rgMT)的三维结构建模

水稻类金属硫蛋白(rgMT)的两端是高度保守的半胱氨酸富含区的结构域(CR区),中间是不含半胱氨酸的间隔区,呈典型的三段式结构.本研究分别采用距离几何算法和同源建模相结合的方法对水稻类金属硫蛋白进行三级结构建模.在排列出CR区的所有可能的半胱氨酸-金属硫络合的组合方式,并对每一种组合方式给出一定的限制条件后各生成20个随机构象.根据生成的随机构象是否能形成金属硫络合结构,从900个随机构象中最终选出6个构象(N端4种,C端2种组合)作为可能的结构模型.另一方面,采用GOR方法对间隔区进行了二级结构预测,随后用同源建模法对其建模.将上述建成的三部分模型连接起来后形成rgMT的整体三维构象.结果表明rgMT能像哺乳动物MT蛋白一样,可形成两个独立的、在结构和能量上均没有障碍的金属-硫络合结构.介于所有植物类金属硫蛋白都具有典型的三段式结构,其中的一部分还具有与rgMT相同的半胱氨酸排列方式,所以rgMT三维结构模型的建立对于其他植物类金属硫蛋白的结构研究具有重要的参考价值.     

A tribute to sulfur.

Recent progress in a number of areas of biochemistry and biology has drawn attention to the critical importance of sulfur in the biosynthesis of vital cofactors and active sites in proteins, and in the complex reaction mechanisms often involved. This brief review is intended as a broad overview of this currently rapidly moving field of sulfur biochemistry, for those who are interested or are involved in one or the other aspect of it, a synopsis by one who has stumbled into this field from several directions in the course of time. Only for iron are metal-sulfur relationships discussed in detail, as the iron-sulfur subfield is one of the most active areas.     

π Delocalization in iridathiabenzenes

Fenske-Hall calculations were carried out for (PEt₃)₃Ir(C₇H₉) (1), [(PEt₃)₃Ir(C₆H₈S)]⁺ (2), [(S-t-but)(PEt₃)₂]Ir(C₆H₈S) (3), and [(S-t-but)(PMet₃)₃]Ir(C₆H₈S) (4) in order to compare the degree of π delocalization in the metallathiacycle rings of (2) and (3). In comparison to (1), a true iridabenzene and (4), an iridathiacyclobutadiene, the π ring systems in (2) and (3) are considerably more localized than the π system in (1) but are not totally localized. Strong metal-sulfur bonding in (2) disrupts the π ring system and results in some localization of the ring π bonds. The introduction of the donor thiolate ligand in (3) disrupts the ring of π system even more by destabilizing the metal orbitals used for metal-sulfur interactions. This weakens the metal-sulfur interaction seen in (2) and leads to even more localization of the ring π system in (3).     

MOSC domains: ancient, predicted sulfur-carrier domains, present in diverse metal-sulfur cluster biosynthesis proteins including Molybdenum cofactor sulfurases

Using computational analysis, a novel superfamily of beta-strand-rich domains was identified in the Molybdenum cofactor sulfurase and several other proteins from both prokaryotes and eukaryotes. These MOSC domains contain an absolutely conserved cysteine and occur either as stand-alone forms such as the bacterial YiiM proteins, or fused to other domains such as a NifS-like catalytic domain in Molybdenum cofactor sulfurase. The MOSC domain is predicted to be a sulfur-carrier domain that receives sulfur abstracted by the pyridoxal phosphate-dependent NifS-like enzymes, on its conserved cysteine, and delivers it for the formation of diverse sulfur-metal clusters. The identification of this domain may clarify the mechanism of biogenesis of various metallo-enzymes including Molybdenum cofactor-containing enzymes that are compromised in human type II xanthinuria.     

Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of [Cd7]-metallothionein-2 from rat liver was determined in aqueous solution, using nuclear magnetic resonance spectrometry and distance geometry calculations. The experimental data provided proton-proton distance constraints from measurements of nuclear Overhauser effects, constraints on the geometry of the metal-cysteine clusters determined by heteronuclear correlation spectroscopy, and dihedral angle constraints derived from both coupling constants and nuclear Overhauser effects. The structure calculations were performed with the program DISMAN. As in previous studies with rabbit liver metallothionein-2a, the structure calculations were performed separately for the alpha and beta-domains containing the 4 and 3-metal clusters, respectively, since no interdomain constraints were found. For both domains, the global polypeptide fold, the location of polypeptide secondary structure elements, the architecture of the metal-sulfur cluster and the local chirality of the metal co-ordination are very similar to the solution structure of rabbit metallothionein-2a, but show considerable difference relative to the crystal structure of rat metallothionein-2.     

Histidine ligands in bacterial metallothionein enhance cluster stability

The cyanobacterial metallothionein (MT) SmtA is the prototype for bacterial MTs and protects against elevated levels of zinc. In contrast to mammalian MTs, bacterial MTs coordinate to metal ions not only via cysteine sulfurs, but unusually for MTs, also via histidine nitrogens. To investigate whether histidine coordination in these metal-sulfur clusters provides advantages over S-coordination only, we mutated the two metal-binding histidine residues in the cyanobacterial MT SmtA from Synechococcus PCC7942 to cysteines. We show that the mutant proteins are still capable of binding up to four zinc ions as is the wild-type protein. However, the mutations perturb protein folding and metal-binding dynamics. Interestingly, several homologues of SmtA also show variations in these two residues. We conclude that histidine residues in Synechococcus PCC7942 SmtA have a stabilising effect due to electrostatic interactions that impact on protein folding and metal cluster charge, and are involved in fine-tuning the reactivity of the bound metal ions.     

Thermodynamic properties of oxidoreductase, transferase, hydrolase, and ligase reactions

Transferases formally couple together two oxidoreductase reactions or two hydrolase reactions. Therefore the thermodynamic properties of transferase reactions can be calculated from differences between thermodynamic properties of two oxidoreductase or two hydrolase reactions. Ligases couple together two hydrolase reactions, and so their thermodynamic properties can be calculated from differences between two hydrolase reactions. These relationships are demonstrated by calculating standard transformed Gibbs energies of reaction and the changes in binding of hydrogen ions at pHs 5-9 of a number of oxidoreductase, transferase, hydrolase, and ligase reactions by use of the data base BasicBiochemData2 and its recent extensions. Coupling is not restricted to two reactions, and an example is given of the coupling of three reactions.     

Heats of reaction of HMo(CO)3(C5R5) (R = H, CH3) with diphenyldisulfide and of formation of the clusters [PhSMo(CO)x(C5H5)]2, X = 1,2. Thermodynamic study of molybdenum-sulfur bond strengths

The enthalpies of reaction of HMo(CO)₃C₅R₅ (R = H, CH₃) with diphenyldisulfide producing PhSMo(CO)₃C₅R₅ and PhSH have been measured in toluene and THF solution (R = H, ΔH= −8.5 ± 0.5 kcal mol⁻¹ (tol), −10.8 ± 0.7 kcal mol⁻¹ (THF); R = CH₃, ΔH = −11.3±0.3 kcal mol⁻¹ (tol), −13.2±0.7 kcal mol⁻¹ (THF)). These data are used to estimate the Mo---SPh bond strength to be on the order of 38–41 kcal mol⁻¹ for these complexes. The increased exothermicity of oxidative addition of disulfide in THF versus toluene is attributed to hydrogen bonding between thiophenol produced in the reaction and THF. This was confirmed by measurement of the heat of solution of thiophenol in toluene and THF. Differential scanning calorimetry as well as high temperature calorimetry have been performed on the dimerization and subsequent decarbonylation reactions of PhSMo(CO)₃Cp yielding [PhSMo(CO)₂Cp]₂ and [PhSMo(CO)Cp]₂. The enthalpies of reaction of PhSMo(CO)₃Cp and [PhSMo(CO)₂Cp]₂ with PPh₃, PPh₂Me and P(OMe)₃ have also been measured. The disproportionation reaction: 2[PhSMo(CO)₂Cp]₂ → 2PhSMo(CO)₃Cp + [PhSMP(CO)Cp]₂ is reported and its enthalpy has also been measured. These data allow determination of the enthalpy of formation of the metal-sulfur clusters [PhSMo(CO)_nC₅H₅]₂, N = 1,2.     

Electron transfer in nitrogenase catalysis

Nitrogenase is a two-component enzyme that catalyzes the nucleotide-dependent reduction of N2 to 2NH3. This process involves three redox-active metal-containing cofactors including a [4Fe-4S] cluster, an eight-iron P cluster and a seven-iron plus molybdenum FeMo-cofactor, the site of substrate reduction. A deficit-spending model for electron transfer has recently been proposed that incorporates protein conformational gating that favors uni-directional electron transfer among the metalloclusters for the activation of the substrate-binding site. Also reviewed is a proposal that each of the metal clusters cycles through only two redox states of the metal-sulfur core as the system accumulates the multiple electrons required for substrate binding and reduction. In particular, it was suggested that as FeMo-cofactor acquires the four electrons necessary for optimal binding of N2, each successive pair of electrons is stored as an Fe-H--Fe bridging hydride, with the FeMo-cofactor metal-ion core retaining its resting redox state. We here broaden the discussion of stable intermediates that might form when FeMo-cofactor receives an odd number of electrons.     

Biochemistry of terminal deoxynucleotidyltransferase: identification, characterization, requirements, and active-site involvement in the catalysis of associated pyrophosphate exchange and pyrophosphorolytic activity

Terminal deoxynucleotidyltransferase (TdT) has been found to catalyze both pyrophosphate exchange and pyrophosphorolysis reactions. Both reactions are strongly inhibited by antiserum to TdT. The reactions require the presence of a divalent cation, a single- or double-stranded oligomeric or polymeric DNA or RNA, and deoxyribonucleoside triphosphates (for PPi exchange only). Of the three divalent cations tested, Mg2+ and Co2+ are equally effective, while Mn2+ neither is used for catalysis nor inhibits the Mg2+-catalyzed reactions. Ribonucleoside triphosphates have been found to support the PPi exchange reaction to a minor extent and have no inhibitory effect on the catalysis mediated by dNTPs. Inhibition studies, using SH group inhibitors, Zn chelator, and a substrate binding site specific reagent, revealed that PPi exchange and pyrophosphorolysis reactions may be distinguished by differences in their sensitivity to inhibition by various reagents. While the PPi exchange reaction is strongly inhibited by sulfhydryl reagents, o-phenanthroline, and pyridoxal phosphate, the pyrophosphorolysis reaction is insensitive to these reagents. In addition, the pyrophosphorolysis reaction is also found not to require a free 3'-OH terminus of a primer. This difference in the susceptibility of the two reactions indicates that discrete active-site structures exist in TdT which catalyze PPi exchange and pyrophosphorolysis reactions.     

Metal substitution of Neurospora copper metallothionein

The binding of diamagnetic Zn(II), Cd(II), and Hg(II) and paramagnetic Co(II) and Ni(II) ions to the apo form of Neurospora metallothionein (MT) was investigated by various spectroscopic techniques. In contrast to native copper MT, which was shown to bind 6 mol of Cu(I)/mol of protein (Lerch, 1980), all substituted forms reveal an overall metal to protein stoichiometry of 3. The charge-transfer (CT) transitions of the complexes containing diamagnetic metal ions as well as the d-d transitions of those with paramagnetic metal ions are indicative of a distorted Td coordination. Electron paramagnetic resonance and absorption measurements of the Co(II) derivative are in agreement with the presence of a metal-thiolate cluster in this protein. Metal titration studies of the apoprotein reveal characteristic spectral features for the derivatives containing two metal equivalents as compared to those with a full complement of three metal ions. The former features are indicative of an exclusive Td type of metal-sulfur coordination whereas the latter suggest that the third metal ion is coordinated in a different fashion. This finding is in agreement with the presence of only seven cysteine residues in Neurospora MT as opposed to nine cysteine residues in the three-metal cluster of the mammalian MT's [Winge, D.R., & Miklossy, K.-A. (1982) J. Biol. Chem. 257, 3471].     

Stoichiometric design of metabolic networks: multifunctionality,clusters, optimization,weak and strong robustness

Starting from a limited set of reactions describing changes in the carbon skeleton of biochemical compounds complete sets of metabolic networks are constructed. The networks are characterized by the number and types of participating reactions. Elementary networks are defined by the condition that a specific chemical conversion can be performed by a set of given reactions and that this ability will be lost by elimination of any of these reactions. Groups of networks are identified with respect to their ability to perform a certain number of metabolic conversions in an elementary way which are called the network’s functions. The number of the network functions defines the degree of multifunctionality. Transitions between networks and mutations of networks are defined by exchanges of single reactions. Different mutations exist such as gain or loss of function mutations and neutral mutations. Based on these mutations neighbourhood relations between networks are established which are described in a graph theoretical way. Basic properties of these graphs are determined such as diameter, connectedness, distance distribution of pairs of vertices. A concept is developed to quantify the robustness of networks against changes in their stoichiometry where we distinguish between strong and weak robustness. Evolutionary algorithms are applied to study the development of network populations under constant and time dependent environmental conditions. It is shown that the populations evolve toward clusters of networks performing a common function and which are closely neighboured. Under changing environmental conditions multifunctional networks prove to be optimal and will be selected.     

Expanding Metabolic Networks: Scopes of Compounds, Robustness, and Evolution

A new method for the mathematical analysis of large metabolic networks is presented. Based on the fact that the occurrence of a metabolic reaction generally requires the existence of other reactions providing its substrates, series of metabolic networks are constructed. In each step of the corresponding expansion process those reactions are incorporated whose substrates are made available by the networks of the previous generations. The method is applied to the set of all metabolic reactions included in the KEGG database. Starting with one or more seed compounds, the expansion results in a final network whose compounds define the scope of the seed. Scopes of all metabolic compounds are calculated and it is shown that large parts of cellular metabolism can be considered as the combined scope of simple building blocks. Analyses of various expansion processes reveal crucial metabolites whose incorporation allows for the increase in network complexity. Among these metabolites are common cofactors such as NAD⁺, ATP, and coenzyme A. We demonstrate that the outcome of network expansion is in general very robust against elimination of single or few reactions. There exist, however, crucial reactions whose elimination results in a dramatic reduction of scope sizes. It is hypothesized that the expansion process displays characteristics of the evolution of metabolism such as the temporal order of the emergence of metabolic pathways. [Reviewing Editor : Dr. David Pollock]