Control of metalloprotein reduction potential: compensation phenomena in the reduction thermodynamics of blue copper proteins

The reduction thermodynamics (Delta H degrees '(rc) and Delta S degrees '(rc)) for native Paracoccus versutus amicyanin, for Alcaligenes faecalis S-6 pseudoazurin, and for the G45P, M64E, and K27C variants of Pseudomonas aeruginosa azurin were measured electrochemically. Comparison with the data available for other native and mutated blue copper proteins indicates that the features of metal coordination and the electrostatic potential due to the protein matrix and the solvent control the reduction enthalpy in a straightforward way. However, the effects on the reduction potential are rather unpredictable owing to the entropic contribution to E degrees ', which is mainly determined by solvent reorganization effects. Analysis of all the Delta H degrees '(rc) and Delta S degrees '(rc) values available for this protein class indicates that enthalpy-entropy compensation occurs in the reduction thermodynamics of wt cupredoxins from different sources, as well as for mutants of the same species. The findings indicate that the reduction enthalpies and entropies for these species are strongly affected by reduction-induced reorganization of solvent molecules within the solvation sphere of the protein. The absence of a perfect enthalpy-entropy compensation is due to the fact that while the differences between reduction entropies are dominated by solvent reorganization effects, those between reduction enthalpies are significantly controlled by intrinsic molecular factors related to the selective stabilization of the reduced form by coordination features of the copper site and electrostatic effects at the interface with the protein matrix.     

Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for blue-copper proteins

 The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques in different conditions of temperature, pH and ionic composition of the medium. The most notable properties of CBP include a positive entropy change upon reduction, a low-pH protonation and detachment of a metal-binding histidine in the reduced protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to some extent the redox thermodynamics. The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations. This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein class. It emerges, in line with previous studies of the electronic structure of blue-copper sites, that the enthalpic contribution to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N₂S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts for the ionic strength dependence of the reduction potential. Received: 20 December 1996 / Accepted: 26 March 1997     

Redox reactivity of the heme Fe<Superscript>3+</Superscript>/Fe<Superscript>2+</Superscript> couple in native myoglobins and mutants with peroxidase-like activity

The reaction enthalpy and entropy for the one-electron reduction of the ferric heme in horse heart and sperm whale aquometmyoglobins (Mb) have been determined exploiting a spectroelectrochemical approach. Also investigated were the T67R, T67K, T67R/S92D and T67R/S92D Mb-H variants (the latter containing a protoheme-l-histidine methyl ester) of sperm whale Mb, which feature peroxidase-like activity. The reduction potential (E°′) in all species consists of an enthalpic term which disfavors Fe³⁺ reduction and a larger entropic contribution which instead selectively stabilizes the reduced form. This behavior differs from that of the heme redox enzymes and electron transport proteins investigated so far. The reduction thermodynamics in the series of sperm whale Mb variants show an almost perfect enthalpy–entropy compensation, indicating that the mutation-induced changes in are dominated by reduction-induced solvent reorganization effects. The modest changes in E°′ originate from the enthalpic effects of the electrostatic interactions of the heme with the engineered charged residues. The small influence that the mutations exert on the reduction potential of myoglobin suggests that the increased peroxidase activity of the variants is not related to changes in the redox reactivity of the heme iron, but are likely related to a more favored substrate orientation within the distal heme cavity.     

Unifying mechanism for metals in toxicity,carcinogenicity and therapeutic action: integrated approach involving electron transfer,oxidative stress,antioxidants, cell signaling and receptors

This comprehensive review of biometals involves the unifying theme of electron transfer, reactive oxygen species, and oxidative stress (OS) applied to toxicity, carcinogenicity, and therapeutic action. The beneficial effect of antioxidants supports the participation of OS. The metals involved are mainly in the heavier category. An important aspect is the favorable reduction potential exhibited by the bioactive materials that permits redox cycling in vivo with the generation of oxy radicals. The basic mechanistic theme is applicable to other electron transfer (ET) functionalities. Appreciable evidence indicates the participation of cell signaling in various ways. Also, a simplifying framework is provided based on radical species and electrochemistry (ET and molecular electrostatic potential). This review also discusses receptor participation with focus on binding to proteins. Resultant physiological effects are summarized. The overview provides an integrated approach to metal bioactivation.     

Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model.

The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.     

钼铁蛋白铁钼辅因子的有机组分对其功能的影响

棕色固氮菌(Azotobacter vinelandii)固氮酶的钼铁蛋白经邻菲啰啉在厌氧或有氧环境中处理后,变为 P-cluster 单一缺失或 P-cluster 和 FeMoco 同时缺失的失活钼铁蛋白。含柠檬酸盐或高柠檬酸盐的重组液都使这两种失活蛋白能恢复固氮酶重组的 H~ 和 C_2H_2还原活性,活性恢复程度随反映钼铁蛋白中金属原子簇含量变化的圆二色和磁圆二色谱及金属含量的恢复程度的提高而提高,但它们固 N_2能力的恢复程度则不相同:P-cluster 单一缺失的蛋白用两种重组液重组后均可恢复其固 N_2能力,而 P-cluster 和 FeMoco 同时缺失的蛋白,只有用含高柠檬酸盐的重组液重组才恢复其固 N_2能力,表明含不同有机组分的重组液所组装的 P-cluster 均与天然状态相同,只有含高柠檬酸盐的重组液所组装的 FeMoco 才与天然状态相同,从而证明高柠檬酸盐是 FeMoco 的必需的有机组分。     

Electron transfer within nitrogenase: evidence for a deficit-spending mechanism

The reduction of substrates catalyzed by nitrogenase utilizes an electron transfer (ET) chain comprised of three metalloclusters distributed between the two component proteins, designated as the Fe protein and the MoFe protein. The flow of electrons through these three metalloclusters involves ET from the [4Fe-4S] cluster located within the Fe protein to an [8Fe-7S] cluster, called the P cluster, located within the MoFe protein and ET from the P cluster to the active site [7Fe-9S-X-Mo-homocitrate] cluster called FeMo-cofactor, also located within the MoFe protein. The order of these two electron transfer events, the relevant oxidation states of the P-cluster, and the role(s) of ATP, which is obligatory for ET, remain unknown. In the present work, the electron transfer process was examined by stopped-flow spectrophotometry using the wild-type MoFe protein and two variant MoFe proteins, one having the β-188(Ser) residue substituted by cysteine and the other having the β-153(Cys) residue deleted. The data support a "deficit-spending" model of electron transfer where the first event (rate constant 168 s(-1)) is ET from the P cluster to FeMo-cofactor and the second, "backfill", event is fast ET (rate constant >1700 s(-1)) from the Fe protein [4Fe-4S] cluster to the oxidized P cluster. Changes in osmotic pressure reveal that the first electron transfer is conformationally gated, whereas the second is not. The data for the β-153(Cys) deletion MoFe protein variant provide an argument against an alternative two-step "hopping" ET model that reverses the two ET steps, with the Fe protein first transferring an electron to the P cluster, which in turn transfers an electron to FeMo-cofactor. The roles for ATP binding and hydrolysis in controlling the ET reactions were examined using βγ-methylene-ATP as a prehydrolysis ATP analogue and ADP + AlF(4)(-) as a posthydrolysis analogue (a mimic of ADP + P(i)).     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

Characterization of Electron Tunneling and Hole Hopping Reactions between Different Forms of MauG and Methylamine Dehydrogenase within a Natural Protein Complex

Respiration, photosynthesis, and metabolism require the transfer of electrons through and between proteins over relatively long distances. It is critical that this electron transfer (ET) occur with specificity to avoid cellular damage, and at a rate that is sufficient to support the biological activity. A multistep hole hopping mechanism could, in principle, enhance the efficiency of long-range ET through proteins as it does in organic semiconductors. To explore this possibility, two different ET reactions that occur over the same distance within the protein complex of the diheme enzyme MauG and different forms of methylamine dehydrogenase (MADH) were subjected to kinetic and thermodynamic analysis. An ET mechanism of single-step direct electron tunneling from diferrous MauG to the quinone form of MADH is consistent with the data. In contrast, the biosynthetic ET from preMADH, which contains incompletely synthesized tryptophan tryptophylquinone, to the bis-Fe(IV) form of MauG is best described by a two-step hole hopping mechanism. Experimentally determined ET distances matched the distances determined from the crystal structure that would be expected for single-step tunneling and multistep hopping. Experimentally determined relative values of electronic coupling (H(AB)) for the two reactions correlated well with the relative H(AB) values predicted from computational analysis of the structure. The rate of the hopping-mediated ET reaction is also 10-fold greater than that of the single-step tunneling reaction despite a smaller overall driving force for the hopping-mediated ET reaction. These data provide insight into how the intervening protein matrix and redox potentials of the electron donor and acceptor determine whether the ET reaction proceeds via single-step tunneling or multistep hopping.     

Protein free energy landscapes remodeled by ligand binding

Glucose/galactose binding protein (GGBP) functions in two different larger systems of proteins used by enteric bacteria for molecular recognition and signaling. Here we report on the thermodynamics of conformational equilibrium distributions of GGBP. Three fluorescence components appear at zero glucose concentration and systematically transition to three components at high glucose concentration. Fluorescence anisotropy correlations, fluorescent lifetimes, thermodynamics, computational structure minimization, and literature work were used to assign the three components as open, closed, and twisted conformations of the protein. The existence of three states at all glucose concentrations indicates that the protein continuously fluctuates about its conformational state space via thermally driven state transitions; glucose biases the populations by reorganizing the free energy profile. These results and their implications are discussed in terms of the two types of specific and nonspecific interactions GGBP has with cytoplasmic membrane proteins.     

佛波酯诱导内皮素和FOS／JUN基因在血管内皮细胞中的表达及AP－1结合活性

佛波酯诱导内皮素和ＦＯＳ／ＪＵＮ基因在血管内皮细胞中的表达及ＡＰ－１结合活性温进坤,魏素珍（河北医学院生化教研室,石家庄０５００１７）张晨晖,姚阿卿,周爱儒,汤健（北京医科大学心血管基础研究所,北京,１０００８３）关键词内皮素基因表达;ＡＰ－１转录因...     

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants

The ability to construct molecular motifs with predictable properties in aqueous solution requires an extensive knowledge of the relationships between structure and energetics. The design of metal binding motifs is currently an area of intense interest in the bioorganic community. To date synthetic motifs designed to bind metal ions lack the remarkable affinities observed in biological systems. To better understand the structural basis of metal ion affinity, we report here the thermodynamics of binding of divalent zinc ions to wild-type and mutant carbonic anhydrases and the interpretation of these parameters in terms of structure. Mutations were made both to the direct His ligand at position 94 and to indirect, or second-shell, ligands Gln-92, Glu-117, and Thr-199. The thermodynamics of ligand binding by several mutant proteins is complicated by the development of a second zinc binding site on mutation; such effects must be considered carefully in the interpretation of thermodynamic data. In all instances modification of the protein produces a complex series of changes in both the enthalpy and entropy of ligand binding. In most cases these effects are most readily rationalized in terms of ligand and protein desolvation, rather than in terms of changes in the direct interactions of ligand and protein. Alteration of second-shell ligands, thought to function primarily by orienting the direct ligands, produces profoundly different effects on the enthalpy of binding, depending on the nature of the residue. These results suggest a range of activities for these ligands, contributing both enthalpic and entropic effects to the overall thermodynamics of binding. Together, our results demonstrate the importance of understanding relationships between structure and hydration in the construction of novel ligands and biological polymers.     

金属螯合亲和层析分离蛋白质的研究

金属螯合亲和层析是近20年发展起来的一项新型分离技术。它以配基简单、吸附量大、分离条件温和、通用性强等特点,逐渐成为分离纯化蛋白质等生物工程产品最有效的技术之一。本文从单组分蛋白质入手,考查了pH值、铵离子浓度、不同铵盐等对蛋白质洗脱的影响,并进行了分析。还对不同的金属螯合柱和不同性质蛋白质的洗脱性能进行了研究,比较了不同金属离子与蛋白质亲和力的区别,为实际体系的分离研究打下了基础。     

Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear--an intricate bioinorganic workaround to use different metals for the same reaction

The ferritin-like superfamily comprises of several protein groups that utilize dinuclear metal sites for various functions, from iron storage to challenging oxidations of substrates. Ribonucleotide reductase R2 proteins use the metal site for the generation of a free radical required for the reduction of ribonucleotides to deoxyriboinucleotides, the building blocks of DNA. This ubiquitous and essential reaction has been studied for over four decades and the R2 proteins were, until recently, generally believed to employ the same cofactor and mechanism for radical generation. In this reaction, a stable tyrosyl radical is produced following activation and cleavage of molecular oxygen at a dinuclear iron site in the protein. Discoveries in the last few years have now firmly established that the radical generating reaction is not conserved among the R2 proteins but that different subgroups, that are structurally very similar, instead employ di-manganese or heterodinuclear Mn-Fe cofactors as radical generators. This is remarkable considering that the protein must exercise a strict control over oxygen activation, reactive metal-oxygen intermediate species and the resulting redox potential of the produced radical equivalent. Given the differences in redox properties between Mn and Fe, use of a different metal for this reaction requires associated adaptations of the R2 protein scaffold and the activation mechanism. Further analysis of the differences in protein sequence between R2 subgroups have also led to the discovery of new groups of R2-like proteins with completely different functions, expanding the chemical repertoire of the ferritin-like superfamily. This review describes the discoveries leading up to the identification of the different Mn-containing R2 protein groups and our current understanding of them. Hypotheses regarding the biochemical rationale to develop these chemically complex alternative solutions are also discussed.     

不同强度运动训练对心肌凋亡途径微小RNA及其靶蛋白的影响

目的：考察不同负荷运动训练对小鼠心肌凋亡相关miR-1,miR-21和靶蛋白的影响,探讨运动干预心肌凋亡的可能机制。方法：选取21只C57BL/6小鼠,随机分为3组（n=7）：安静组（SE组）、训练1组（ET1组）、训练2组（ET2）。SE组不进行训练,ET1组完成8周递增负荷游泳训练,5天/周,1次/天,第1周30 min/count,每周增加10 min,第7、8周时间维持在90 min;ET2组在ET1组方案基础上增加负荷,前5周与ET1相同,后3周每天训练2次。TUNEL检测考察心肌凋亡水平,Western blot和RT-PCR分别测定蛋白和miRs的变化。结果：ET1组游泳训练对小鼠心肌凋亡影响不明显,miR-1表达无显著变化,但其靶蛋白Bcl-2表达显著增高（P<0.01）,miR-21及其靶蛋白PDCD4表达均无显著变化。ET2组游泳训练显著降低心肌凋亡水平及miR-1表达（P<0.01）、提高Bcl-2表达（P<0.05）;同时显著提高miR-21表达（P<0.05）,但对PDCD4表达无明显影响。结论：ET1组训练对心肌凋亡干预不明显,ET2组运动训练可降低心肌凋亡水平,miR-1及靶蛋白Bcl-2变化可能是机制之一,PDCD4对运动训练不敏感,miR-21可能与其它靶蛋白参与运动干预心肌凋亡的分子机制。     

Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.     

Interaction of phospholipid-metal complexes with water-soluble wheat protein

Insoluble lipid-protein complexes are formed in the presence of Ni(II), Ca(II), or Mg(II) by specific components of the water-soluble proteins of wheat flour and either triphosphoinositide or phosphatidyl serine. The pattern of protein species bound by the lipid-metal complex is dependent upon the metal and the phospholipid used. A group of proteins, containing carbohydrate, may be solubilized and recovered by washing the precipitate with acidic chloroform-methanol-water. Analyses of reactive and nonreactive protein species have shown no differences which clearly account for their behavior. Methylation of protein increases binding to lipid; acetylation decreases the interaction. Weak interaction has been observed between certain components of flour proteins and phospholipid in the absence of metal ions, but the components differ from those bound in the presence of metal ions. It is suggested that properly oriented groups of the protein molecules are chelating onto available coordination positions of metal ions already bound to phospholipid.     

The molecular characteristics of two metallothioneins in Ostrinia furnacalis and expression under conditions of heavy metal stress

Metallothioneins are ubiquitously-expressed metal-binding proteins. Despite their potential ecological relevance, no prior reports have identified any metallothioneins in Ostrinia furnacalis or other Lepidoptera species. A better understanding of the molecular characteristics and regulatory dynamics of metallothionein genes in O. furnacalis under heavy metal stress conditions would enable future studies of the roles played by these proteins in the context of heavy metal detoxification. Herein, we identified and characterized two metallothionein (OfMT) genes in O. furnacalis, including the 147 bp OfMT1 gene encoding a 48 amino acid protein containing 10 cysteine residues, and the 141 bp OfMT2 gene encoding a 46 amino acid protein containing 12 cysteine residues. The expression of OfMT2 was found to be related to Cu and Cd concentrations in a dose-dependent manner but was unaffected by Zn exposure. Overall, these results indicate that OfMT genes likely encode metal-binding proteins consistent with their potential role in the maintenance of heavy metal homeostasis.     

Redox thermodynamics of the ferric-ferrous couple of wild-type synechocystis KatG and KatG(Y249F)

Crystal structures and mass spectrometric analyses of catalase-peroxidases (KatGs) from different organisms revealed the existence of a peculiar distal Met-Tyr-Trp cross-link. The adduct appears to be important for the catalase but not the peroxidase activity of bifunctional KatG. To examine the effect of the adduct on enzyme redox properties and functions, we have determined the thermodynamics of ferric reduction for wild-type KatG and KatG(Y249F), whose tyrosine-to-phenylalanine mutation prevents cross-link formation. At 25 degrees C and pH 7.0, the reduction potential of wild-type KatG is found to be -226 +/- 10 mV, remarkably lower than the published literature values. The reduction potential of KatG(Y249F) is very similar (-222 +/- 10 mV), but variable temperature experiments revealed compensatory differences in reduction enthalpies and entropies. In both proteins, the oxidized state is enthalpically stabilized over the reduced state, but entropy is lost on reduction, which is in strong contrast to horseradish peroxidase, which also features a much more pronounced enthalpic stabilization of the ferriheme. With both proteins, the midpoint potential increased linearly with decreasing pH. We discuss whether the observed redox thermodynamics reflects the differences in structure and function between bifunctional KatG and monofunctional peroxidases.