Tandem mass spectrometry of protein—Protein complexes: Cytochrome c—Cytochrome b 5

An improved method to interpret triple quadrupole MS/MS experiments of complexes of large ions is presented and applied to a study of the complex formed by the proteins cytochrome c and cytochrome b5. Modeling of the activation and dissociation process shows that most of the reaction occurs near the collision cell exit where ions have the highest internal energies. Experiments at different collision cell pressures or with different collision gases (Ne, Ar, Kr) are interpreted with a previously proposed collision model (Chen et al., Rapid Commun. Mass Spectrom. 1998, 12, 1003-1010) to calculate the internal energy added to ions to cause dissociation. Small but systematic differences under different experimental conditions are attributed to different times available for reaction. A method to correct for this is presented. Ne, Ar, and Kr are found to have similar energy transfer efficiencies. Complexes of cytochrome c and cytochrome b5 are detected in ESI mass spectra but with abundances less than expected from the solution equilibrium. Dissociation of the cytochrome c-cytochrome b5 complexes with charge k gives as the most abundant fragments, cytochrome b5(+3) and cytochrome c+(k-3). Adding charges to the complex destabilizes it. A series of cytochrome c variants with Lys residues thought to be involved in solution binding replaced by Ala showed no differences in the energy required to induce dissociation of the gas phase complex. The implications for the binding of the gas phase ions are inconclusive.     

表面带负电荷氨基酸残基的突变对细胞色素b5与细胞色素c结合和识别的影响

利用核磁共振方法研究表面带不同负电荷氨基酸残基突变后的细胞色素b₅与细胞色素c的结合与识别.结果表明,静电作用在细胞色素b₅与细胞色素c的结合过程中有着重要的贡献,而且这些静电贡献在一定程度上具有累加性,E48的贡献略大于E44.同时还证明Browniandynamicssimulations优化出的Glu48-Lys13,Glu56-Lys₈₇,Asp60-Lys86和heme6-propionate-Tml₇₂(细胞色素b₅的残基排在前面)的结合方式在溶液中的确存在.细胞色素b₅突变体(E48,E56/A,D60/A)及[Cr(oxalate)₃]^3-对细胞色素c的表面结合竞争实验表明,细胞色素c表面结合区Site仍然同细胞色素b₅突变体(E48,E56/A,D60/A)有结合作用,只是结合强度上相对于野生细胞色素b₅同细胞色素c的结合有所降低.这表明除上述的Brownian dynamics simulations模型外,尚有其它如Salemme模型等的结合方式,这也揭示出细胞色素b₅和细胞色素c之间的结合是比较动态的.     

Study on the Gas Phase Stability of Heme-binding Pocket in Cytochrome Tb_5 and Its Mutants by Electrospray Mass Spectrometry

Ｃｙｔｏｃｈｒｏｍｅｂ5(Ｃｙｔｂ5)ｉｓｆｏｕｎｄｂｏｔｈａｓａｃｏｍｐｏ ｎｅｎｔｏｆｔｈｅｍｉｃｒｏｓｏｍａｌｍｅｍｂｒａｎｅｓａｎｄａｓａｓｏｌｕｂｌｅｆｏｒｍｉｎｅｒｙｔｈｒｏｃｙｔｅｓ .Ｉｔｐｌａｙｓａｎｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｂｉｏｌｏｇｉｃａｌｓｙｓｔｅｍｓ ,ｉｎｗｈｉｃｈＣｙｔｂ5ｆｕｎｃｔｉｏｎｓａｓａｎｅｌｅｃｔｒｏｎｃａｒｒｉｅｒ,ｐａｒｔｉｃｉｐａｔｉｎｇｉｎａｓｅｒｉｅｓｏｆｅｌｅｃｔｒｏｎ ｔｒａｎｓｆｅｒｐｒｏｃｅｓｓｅｓ ,ｉｎ ｃｌｕｄｉｎｇｒｅｄｕｃｔｉｏｎｏｆ…     

Influences of the Hydrophobicity of the Heme－binding Pocket on the Properties and Functions of Cytochrome b5 Mutants

The mutation sites of the four mutants F35Y, P40V, V45E and V45Y of cytochrome b5 are located at the edge of the heme-binding pocket. The solvent accessible areas of the “pocket inte-rior“ of the four mutants and the wild-type cytochrome b5 have been calculated based on their crystal structures at high resolu-tion. The change in the hydrophobicity of the heme-binding pocket resulting from the mutation can be quantitatively de-scribed using the difference of the solvent accessible area of the “pocket interior“ of each mutant from that of the wild-type cy-tochrome b5. The influences of the hydrophobicity of the heme-binding pocket on the protein stability and redox potential are discussed.     

细胞色素c突变体的共振拉曼光谱研究

采用共振拉曼光谱技术研究了细胞色素c一次突变体（WT）及其突变体Y67F和N52I在低频区的光谱特征。结果表明，以苯丙氨酸替代WT中酪氨酸残基Tyr67并没有明显影响血红素丙氨酸侧基周围多肽氨基酸残基的构象，而异亮氨酸对天冬酰胺残基Asn52的取代则较大程度地改变了蛋白质内部水分子与周围氨基酸残基间的氢键作用和多肽空腔的疏水性，进而使氨基酸残基和血素的构象相应发生调变。两种取代都导致形成血红素周围空腔的多肽氨基酸残基构象的变化。     

Cytochrome c(2) Exit Strategy: Dissociation Studies and Evolutionary Implications

Small, water-soluble, type c cytochromes form a transient network connecting major bioenergetic membrane protein complexes in both photosynthesis and respiration. In the photosynthesis cycle of Rhodobacter sphaeroides, cytochrome c2 (cyt c2) docks to the reaction center (RC), undergoes electron transfer, and exits for the cytochrome bc1 complex. Translations of cyt c2 about the RC-cyt c2 docking interface and surrounding membrane reveal possible exit pathways. A pathway at a minimal elevation allowed by the architecture of the RC is analyzed using both an all-atom steered molecular dynamics simulation of the RC-cyt c2 complex and a bioinformatic analysis of the structures and sequences of cyt c. The structure-based phylogenetic analysis allows for the identification of structural elements that have evolved to satisfy the requirements of having multiple functional partners. The patterns of evolutionary variation obtained from the phylogenetic analysis of both docking partners of cyt c2 reveal conservation of key residues involved in the interaction interfaces that would be candidates for further experimental studies. Additionally, using the molecular mechanics Poisson-Boltzmann surface area method we calculate that the binding free energy of reduced cyt c2 to the RC is nearly 6 kcal/mol more favorable than with oxidized cyt c2. The redox-dependent variations lead to changes in structural flexibility, behavior of the interfacial water molecules, and eventually changes in the binding free energy of the complex.     

牛视紫红质蛋白质中视黄醛的活性位点

视紫红质蛋白是一个跨膜蛋白, 视黄醛(RET)在该蛋白中的活性结合位点涉及到视觉过程机理, 与一些眼科疾病病理有关. 基于牛视紫红质蛋白1U19的蛋白质晶体结构数据, 采用密度泛函理论的B3LYP方法计算RET-Lys296残基与视黄醛分子周围半径为0.6 nm的空间范围30个氨基酸残基相互作用和结合能. 数值显示1U19蛋白中的残基Glu113、Glu181和Glu122是质子化的RET-Lys296残基的活性结合位点, 结合能分别为-333.38、-205.67和-194.56 kJ·mol-1. 这些氨基酸残基带有一个负电荷, 与质子化的RET-Lys296残基发生强烈的离子静电相互作用. 另外几个残基Ala292、Cys187、Phe293、Pro291以及Trp265等与质子化RET-Lys296残基也有相互吸引作用. 当RET-Lys296残基非质子化, 上述相互作用消失, 促使视黄醛分子与视蛋白分离. 研究发现残基Glu113和Glu181周围各自有一个结晶水分子通过双氢键形式起着稳定作用.     

Investigating the Active Oxidants Involved in Cytochrome P450 Catalyzed Sulfoxidation Reactions

Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C−H bonds of organic molecules. This reaction is initiated by a ferryl-oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric-hydroperoxy complex (Cpd 0) and the Fe(III)-H₂O₂ complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4-alkylthiobenzoic acids and 4-methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol-pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X-ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O-demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O-demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O-dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)-H₂O₂ complex which would be more abundant in the peroxide-driven reactions.     

Modification of <Emphasis Type="Italic">Cytochrome c</Emphasis> by 4-hydroxy- 2-nonenal: Evidence for histidine,lysine, and arginine-aldehyde adducts

4-Hydroxy-2-nonenal (4HNE), a major secondary product of lipid peroxidation, has been associated with a number of disease states involving oxidative stress. Despite the recognized importance of post-translational modification of proteins by products such as 4HNE, little is known of the modification of cytochrome c by this reagent and its analysis by mass spectrometry. The purpose of this study was to investigate the chemical interaction of 4HNE and cytochrome c, a protein essential to cellular respiration, under in vitro conditions. Isoelectric focusing of native and 4HNE-modified cytochrome c using immobilized pH gradient (IpG) strips showed a decrease in the pI of the 4HNE-modified protein suggesting modification of charged amino acids. Reaction of 4HNE with cytochrome c resulted in increases in molecular weight consistent with the addition of four 4HNE residues as determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS). Samples of both native and 4HNE-modified cytochrome c were enzymatically digested and subjected to peptide mass fingerprinting using MALDI-TOF MS. Analysis of these samples using LC-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) provided sequence information that was used to determine specific residues to which the aldehyde adducted. Taken together, the data indicated that H33, K87, and R38 were modified by 4HNE. Mapping these results onto the X-ray crystal structure of native cytochrome c suggest that 4HNE adduction to cytochrome c could have significant effects on tertiary structure, electron transport, and ultimately, mitochondrial dysfunction.     

Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam

Oxygenated derivatives of the monoterpene (+)-alpha-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-alpha-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450(cam) for the selective oxidation of (+)-alpha-pinene. Consideration of the structures of (+)-camphor and (+)-alpha-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-alpha-pinene. Some mutants had tighter (+)-alpha-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-alpha-pinene oxidation rate of 270 nmol (nmol of P450(cam))(-)(1) min(-)(1), which was 70% of the rate of camphor oxidation by wild-type P450(cam). Camphor is oxidized by wild-type P450(cam) exclusively to 5-exo-hydroxycamphor. If the gem dimethyl groups of (+)-alpha-pinene occupied similar positions to those found for camphor in the wild-type structure, (+)-cis-verbenol would be the dominant product. All P450(cam) enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared to camphor oxidation by the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-alpha-pinene epoxides were among the minor products. The crystal structure of the Y96F/F87W/V247L mutant, the most selective of the P450(cam) mutants initially examined, was determined to provide further insight into P450(cam) substrate binding and catalysis. (+)-alpha-Pinene was bound in two orientations which were related by rotation of the molecule. One orientation was similar to that of camphor in the wild-type enzyme while the other was significantly different. Analysis of the enzyme/substrate contacts suggested rationalizations of the product distribution. In particular competition rather than cooperativity between the F87W and V247L mutations and substrate movement during catalysis were proposed to be major factors. The crystal structure lead to the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the binding orientation toward that of camphor in the wild-type structure. The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone. The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450(cam) mutant that could oxidize (+)-alpha-pinene directly to (+)-verbenone. Verbenol, verbenone, and myrtenol are naturally occurring plant fragrance and flavorings. The preparation of these compounds by selective enzymatic oxidation of (+)-alpha-pinene, which is readily available in large quantities, could have applications in synthesis. The results also show that the protein engineering of P450(cam) for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme-substrate interactions.     

An evaluation of molecular models of the cytochrome P450 Streptomyces griseolus enzymes P450SU1 and P450SU2

Summary P450SU1 and P450SU2 are herbicide-inducible bacterial cytochrome P450 enzymes from Streptomyces griseolus. They have two of the highest sequence identities to camphor hydroxylase (P450cam from Pseudomonas putida), the cytochrome P450 with the first known crystal structure. We have built several models of these two proteins to investigate the variability in the structures that can occur from using different modeling protocols. We looked at variability due to alignment methods, backbone loop conformations and refinement methods. We have constructed two models for each protein using two alignment algorithms, and then an additional model using an identical alignment but different loop conformations for both buried and surface loops. The alignments used to build the models were created using the Needleman-Wunsch method, adapted for multiple sequences, and a manual method that utilized both a dotmatrix search matrix and the Needleman-Wunsch method. After constructing the initial models, several energy minimization methods were used to explore the variability in the final models caused by the choice of minimization techniques. Features of cytochrome P450cam and the cytochrome P450 superfamily, such as the ferredoxin binding site, the heme binding site and the substrate binding site were used to evaluate the validity of the models. Although the final structures were very similar between the models with different alignments, active-site residues were found to be dependent on the conformations of buried loops and early stages of energy minimization. We show which regions of the active site are the most dependent on the particular methods used, and which parts of the structures seem to be independent of the methods.     

Electrochemical and Spectroscopic Study on the Interaction of Cytochrome c with Anionic Lipid Vesicles

The structure and the electron-transfer of cytochrome c binding on the anionic lipid vesicles wrer analyzed by electrochemical and various spectroscopic methods.It was found that upon binding to anionic lipid membrane,the formal potential of cytochrome c shifted 30 mV negtively indicating an easier redox interaction than that in its native state.This is due to the local alteration of the coordination and the heme crevice.The structural perturbation in which a molten globule-like state is formed during binding to anionic lipid vesicles is more important.This study may help to understand the mechanism of the electron-transfer reactions of cytochrome c at the mitochondrial membrane.     

Studies on Cytochrome c Modified by [PtdienNO_3]Cl

For the first time, [PtdienNO_3]Cl was used as a stable reagent to modify ferricytochrome c and the reaction products were separated and purified with the CM-52 cation exchange chromatography. Five components were obtained, corresponding to the native cytochrome c single-labeled, dual-labeled, and triple-labeled derivatives as shown by the analysis of the molar ratio of the two metal atoms (Pt and Fe). The reduction potentials of these proteins were measured by differential pulse voltammetry. His-33 and Trp-59 were identified by~1HNMR as the binding sites of the platinum complex in the modified cytochrome c derivatives. Trp-59 was a conserved amino acid connected with the heme through hydrogen bond, which had not been modified by other transition metal complexes. The platinummodified cytochrome c derivatives might be valuable in exploring the role of the aromatic amino acids, especially Trp-59, in electron transfer.     

Proline-40 is Essential to Maintaining Cytochrome b_5''''s Stability and Its Electron Transfer with Cytochrome c

ＩｎｔｒｏｄｕｃｔｉｏｎＣｙｔｏｃｈｒｏｍｅｂ5(Ｃｙｔｂ5)ｉｓａｍｅｍｂｒａｎｅ ｂｏｕｎｄｐｒｏ ｔｅｉｎ .Ｉｔｃａｎｂｅｐｒｏｔｅｏｌｙｚｅｄｔｏｙｉｅｌｄａｓｏｌｕｂｌｅ ,ｈｙｄｒｏｐｈｉｌｉｃｄｏｍａｉｎｃｏｎｔａｉｎｉｎｇａｎｏｎ ｃｏｖａｌｅｎｔｌｙｂｏｕｎｄｈｅｍｅｇｒｏｕｐ .Ｃｙｔｂ5ｉｓｉｎｖｏｌｖｅｄｉｎｅｌｅｃｔｒｏｎｔｒａｎｓｆｅｒｗｉｔｈａｖａｒｉｅｔｙｏｆｐｒｏｔｅｉｎｓ,ｓｕｃｈａｓｃｙｔｏｃｈｒｏｍｅｃ (Ｃｙｔｃ) ,1 3 ｍｅｔｍｙｏ ｇｌｏｂｉｎ ,2 ｍｅｔｈｅｍｏｇｌｏ…     

Proteo-dendrimers designed for complementary recognition of cytochrome c: dendrimer architecture toward nanoscale protein complexation

"Proteo-dendrimers" in which polyanionic hepta(glutamic acids), fluorescent zinc porphyrinate cores, hydrophilic polyether surfaces, and nonpeptide hydrophobic dendrons are combined, were developed as a new series of synthetic receptors for protein recognition. They have polyanionic "patch" structures on their surfaces and undergo complementary electrostatic interactions with a positively charged cytochrome c patch, as observed in biological protein-protein recognition systems. Stability constants of the resulting supramolecular complexes were determined in phosphate buffer (pH 7) by monitoring the fluorescence quenching of the zinc porphyrinates. These proteo-dendrimer receptors exhibited higher affinities with cytochrome c proteins in aqueous solutions than with biological cytochrome b5. Furthermore, they effectively blocked complexation of biological cytochrome b5 with cytochrome c, indicating that the proteo-dendrimers and cytochrome b5 similarly occupy the polycationic patch of cytochrome c.     

In silico mutagenesis and docking study of Ralstonia solanacearum RSL lectin: performance of docking software to predict saccharide binding

In this study, in silico mutagenesis and docking in Ralstonia solanacearum lectin (RSL) were carried out, and the ability of several docking software programs to calculate binding affinity was evaluated. In silico mutation of six amino acid residues (Agr17, Glu28, Gly39, Ala40, Trp76, and Trp81) was done, and a total of 114 in silico mutants of RSL were docked with Me-α-L-fucoside. Our results show that polar residues Arg17 and Glu28, as well as nonpolar amino acids Trp76 and Trp81, are crucial for binding. Gly39 may also influence ligand binding because any mutations at this position lead to a change in the binding pocket shape. The Ala40 residue was found to be the most interesting residue for mutagenesis and can affect the selectivity and/or affinity. In general, the docking software used performs better for high affinity binders and fails to place the binding affinities in the correct order.     

Competitive ion-exchange adsorption of proteins: competitive isotherms with controlled competitor concentration

The competitive adsorption processes inevitably present in chromatographic separations of complex mixtures have not been extensively studied. This is partly due to the difficulty of measuring true competitive isotherms, in which all system parameters (including competitor concentrations) are held constant. We report a novel approach to determining competitive protein adsorption isotherms in which the competitor concentration is held constant across the entire isotherm. By using the heme prosthetic group in cytochrome b5 as a quantitative spectrophotometric label, competitive isotherms between cytochrome b5 and alpha-lactalbumin can be constructed. Similarly, manganese-substituted protoporphyrin IX heme replacement allows the non-perturbing labeling of individual cytochrome b5 conservative surface charge mutants by replacement of a single atom in the interior of the protein. This labeling allows the study of competition between cytochrome b5 charge mutants of identical size and shape, which differ only in charge arrangement. Using these techniques, the effect of competing species on equilibrium behavior and the apparent heterogeneity of anion-exchange adsorbents in the presence of competitors can be quantitatively studied by fitting the data to two popular single-component binding models, the Temkin and the Langmuir-Freundlich (L-F) isotherms.     

自组装膜上细胞色素c的电化学石英晶体微天平实时表征和定量检测

采用电化学石英晶体微天平(EQCM)实时表征和定量检测细胞色素c(Cytc).在压电石英晶振表面上自组装巯基十一酸(MUA)单层膜,以盐酸1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)活化羧基,将Cytc共价固化到电极表面.EQCM实时监测了MUA的自组装和Cytc的固化过程,测定了二者在电极表面的覆盖度和Cytc的固化量.结果表明,Cytc在0.03～3.00μmol/L浓度范围内呈线性变化,检测限可达到1.19×10^-9mol/L.     

Why substituting the asparagine at position 35 in Bacillus circulans xylanase with an aspartic acid remarkably improves the enzymatic catalytic activity? A quantum chemistry-based calculation study

Xylanases from Bacillus circulans (BCX) are known as configuration-retaining glycoside hydrolases, which hydrolyze xylans with two glutamic acid residues (Glu78 and Glu172) serving as catalytic active residues according to a double displacement mechanism. Existing experimental researches show that mutating the asparagines (Asn) to aspartic acid (Asp) at position 35 next to Glu172 can obviously improve the catalytic activity of BCX. To better understand the inherent mechanism for the experimental finding, we performed quantum chemistry calculations on two model systems to mimic the catalyses of wild-type and mutant BCXs. Geometrical structures and relative energies of intermediates and transition states involved in the hydrolysis reactions are given in detail. It is found that in the wild-type model system Asn35 interacts with Glu172 via a loose hydrogen bond, while in the mutant model system Asp35 forms a very tight hydrogen bond with Glu172. The glycosidic bond cleavage is proposed to be the rate-determining step for the hydrolysis reaction, whose barrier varies from 98 to 65 kJ mol⁻¹ when Asn35 is replaced by Asp35, showing the presence of Asp35 remarkably reduces the energy demand for the hydrolysis reaction. The present result provides a theoretical elucidation for why a single amino acid substitution can importantly influences catalytic activity of BCX.