Molecular modeling and dynamics simulation of a histidine-tagged cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript>

Although an affinity tag such as six consecutive histidines, (His)₆-tag, has been widely used to obtain high quantity of recombinant proteins, little is known about its influences on heme proteins for lack of structural information. When (His)₆-tag was introduced to the N-terminus of a small heme protein, cytochrome b ₅, experimental results showed the resultant protein, (His)₆-cyt b ₅, has similar property and function to that of isolated cyt b ₅. To provide structural information for this observation, we herein performed a structural prediction of (His)₆-cyt b ₅ by molecular modeling in combination with molecular dynamics simulation. The predicted structure, as assessed by a series of criteria with good quality, reveals that the (His)₆-tag adopts a helical conformation and packs against the hydrophobic core 2 of cyt b ₅ through salt bridges, hydrogen bonding and hydrophobic interactions. The heme group, with the axial His ligands slightly rotated, was found to have similar conformation as in isolated cyt b ₅, which indicates that the N-terminal (His)₆-tag does not alter the heme active site, resulting in similar dynamics properties for core 1. This study provides valuable information of interactions between (His)₆-tag and the rest of the protein, aiding in rational design and application of functional His-tagged proteins.     

Water-splitting manganese complex controls light-induced redox changes of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>559</Subscript> in Photosystem II

The effect of water-splitting Mn complex on light-induced redox changes of cytochrome b ₅₅₉ (cyt b ₅₅₉) was studied in spinach photosystem II (PSII) membranes. Photoreduction of the heme iron in the intact PSII membranes was completely suppressed by DCMU, whereas photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were unaffected by DCMU. Interesingly, photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were completely diminished by exogenous superoxide dismutase (SOD), whereas no effect of SOD on photoreduction of the heme iron was observed in the intact PSII membranes. The current work shows that the light-induced redox changes of cyt b ₅₅₉ proceed via a different mechanism in the both types of PSII membranes. In the intact PSII membranes, photoreduction of the heme iron is mediated by plastoquinol. However, in the Mn-depleted PSII membranes, photoreduction and photooxidation of the heme iron are mediated by superoxide anion radical formed in PSII.     

Proline Rich Polypeptide (PRP-1) Increases the Superoxide-Producing and Ferrihemoglobin Reducing Activities of Cytochrome B<Subscript>558</Subscript> Isoforms from Human Lymphosarcoma Tissue Cells

The two cytochromes (cyt) b₅₅₈ of acidic nature, one—95–100 kDa and another one, 60–70 kDa were isolated for the first time from the human’s lymphosarcoma tissue cells using gel filtration and ion exchange chromatography. These hemoproteins possess NADPH dependent O₂ ⁻-producing and ferrihemoglobin-reducing activities. The incubation of neuropeptide PRP-1 (5 μg) with cytochrome b₅₅₈, caused elevation of these activities. The gel filtration results indicated possible binding of PRP-1 to these cytochromes b₅₅₈. PRP-1 activated both NADPH dependent O₂ ⁻-producing and ferriHb-reducing activities of the cyt b¹ ₅₅₈ and cyt b² ₅₅₈, obtained from human lymphosarcoma tissue cells. One can assume that PRP-1 associated with cyt b₅₅₈ on the surface of the tumor cells by increasing both NADPH dependent O₂ ⁻-producing and ferriHb-reducing activities of cyt b₅₅₈, increases the oxidation- reduction status. Changing the oxidation–reduction status and oxygen homeostasis of the tumor cells by PRP-1 can serve as one of the possible explanation of antitumorigenic effect of this cytokine.     

The structure and function of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>2</Subscript>: reaction center electron transfer complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c₂ (cyt c₂) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c₂ (cyt c₂) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c₂ is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.     

Effect of calcium ions on electron transfer between hemes <Emphasis Type="Italic">a</Emphasis> and <Emphasis Type="Italic">a</Emphasis><Subscript>3</Subscript> in cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH₃) ₆ ³⁺ , three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a ₃ reduction. The difference spectrum of heme a ₃ reduction in the visible region is characterized by a maximum at ～612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a ₃ reduction is slowed down by low concentrations of Ca²⁺. The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca²⁺ ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca²⁺ ions discovered earlier and indicate that the cation affects intramolecular electron transfer.     

Predicting the disruption by of a protein-ligand interaction

The uranyl cation (UO₂²⁺) can be suspected to interfere with the binding of essential metal cations to proteins, underlying some mechanisms of toxicity. A dedicated computational screen was used to identify UO₂²⁺ binding sites within a set of nonredundant protein structures. The list of potential targets was compared to data from a small molecules interaction database to pinpoint specific examples where UO₂²⁺ should be able to bind in the vicinity of an essential cation, and would be likely to affect the function of the corresponding protein. The C‐reactive protein appeared as an interesting hit since its structure involves critical calcium ions in the binding of phosphorylcholine. Biochemical experiments confirmed the predicted binding site for UO₂²⁺ and it was demonstrated by surface plasmon resonance assays that UO₂²⁺ binding to CRP prevents the calcium‐mediated binding of phosphorylcholine. Strikingly, the apparent affinity of UO₂²⁺ for native CRP was almost 100‐fold higher than that of Ca²⁺. This result exemplifies in the case of CRP the capability of our computational tool to predict effective binding sites for UO₂²⁺ in proteins and is a first evidence of calcium substitution by the uranyl cation in a native protein.     

Characterization of two cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript> proteins from the cyanobacterium <Emphasis Type="BoldItalic">Gloeobacter violaceus</Emphasis> PCC 7421

In the genome of the untypical cyanobacterium Gloeobacter violaceus PCC 7421 two potential cytochrome b ₆ proteins PetB1 and PetB2 are encoded. Such a situation has not been observed in cyanobacteria, algae and higher plants before, and both proteins are not characterized at all yet. Here, we show that both apo-proteins bind heme with high affinity and the spectroscopic characteristics of both holo-proteins are distinctive for cytochrome b ₆ proteins. However, while in PetB2 one histidine residue, which corresponds to H100 and serves as an axial ligand for heme b _H in PetB1, is mutated, both PetB proteins bind two heme molecules with different midpoint potentials. To recreate the canonical heme b _H binding cavity in PetB2 we introduced a histidine residue at the position corresponding to H100 in PetB1 and subsequently characterized the generated protein variant. The presented data indicate that two bona fide cytochrome b ₆ proteins are encoded in Gloeobacter violaceus. Furthermore, the two petB genes of Gloeobacter violaceus are each organized in an operon together with a petD gene. Potential causes and consequences of the petB and petD gene heterogeneity are discussed.     

Peculiarities of cyanide binding to the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript>-type cytochrome oxidase from the thermophilic bacterium <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Cytochrome c oxidase of the ba ₃-type from Thermus thermophilus does not interact with cyanide in the oxidized state and acquires the ability to bind heme iron ligands only upon reduction. Cyanide complexes of the reduced heme a ₃ in cytochrome ba ₃ and in mitochondrial aa ₃-type cytochrome oxidase are similar spectroscopically, but the a ₃²⁺-CN complex of cytochrome ba ₃ is strikingly tight. Experiments have shown that the K _d value of the cytochrome ba ₃ complex with cyanide in the presence of reductants of the enzyme binuclear center does not exceed 10⁻⁸ M, which is four to five orders of magnitude less than the K _d of the cyanide complex of the reduced heme a ₃ of mitochondrial cytochrome oxidase. The tightness of the cytochrome ba ₃ complex with cyanide is mainly associated with an extremely slow rate of the ligand dissociation (k _off ≤ 10⁻⁷ sec⁻¹), while the rate of binding (k _on ∼ 10² M⁻¹·sec⁻¹) is similar to the rate observed for the mitochondrial cytochrome oxidase. It is proposed that cyanide dissociation from the cytochrome ba ₃ binuclear center might be hindered sterically by the presence of the second ligand molecule in the coordination sphere of Cu_B²⁺. The rate of cyanide binding with the reduced heme a ₃ does not depend on pH in the neutral area, but it approaches linear dependence on H⁺ activity in the alkaline region. Cyanide binding appears to be controlled by protonation of an enzyme group with pK _a = 8.75.     

P2X<Subscript>7</Subscript> Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X₇ receptor activated a reversible, Ca²⁺-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C_m) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5′-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C_m without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2′,3′-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2′4′-disulfonic acid, adenosine 5′-triphosphate-2′3′-dialdehyde, and Cu²⁺. The capacitance decrease persisted in Na⁺-, Ca²⁺- and Cl⁻-free external saline or with Ca²⁺-free pipette solution. It is concluded that ATP activation of the inotropic P2X₇ receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca²⁺ or influx of Na⁺ or Ca²⁺ through the receptor-gated channel.     

The <Emphasis Type="Italic">b</Emphasis><Subscript>arg36</Subscript> contributes to efficient coupling in F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli, the F₁F_O ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the b _arg36→ile and the b _arg36→glu mutations. The b _arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b _arg36→glu mutation was largely suppressed in the b _{arg36→glu,glu39→arg} double mutant. E. coli expressing the b _{arg36→glu,glu39→arg} subunit grew well on succinate-based medium. F₁F_O ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F₁F_O ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.     

Another look at the interaction between mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> and flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>

Yeast flavocytochrome b ₂ tranfers reducing equivalents from lactate to oxygen via cytochrome c and cytochrome c oxidase. The enzyme catalytic cycle includes FMN reduction by lactate and reoxidation by intramolecular electron transfer to heme b ₂. Each subunit of the soluble tetrameric enzyme consists of an N terminal b ₅-like heme-binding domain and a C terminal flavodehydrogenase. In the crystal structure, FMN and heme are face to face, and appear to be in a suitable orientation and at a suitable distance for exchanging electrons. But in one subunit out of two, the heme domain is disordered and invisible. This raises a central question: is this mobility required for interaction with the physiological acceptor cytochrome c, which only receives electrons from the heme and not from the FMN? The present review summarizes the results of the variety of methods used over the years that shed light on the interactions between the flavin and heme domains and between the enzyme and cytochrome c. The conclusion is that one should consider the interaction between the flavin and heme domains as a transient one, and that the cytochrome c and the flavin domain docking areas on the heme b ₂ domain must overlap at least in part. The heme domain mobility is an essential component of the flavocytochrome b ₂ functioning. In this respect, the enzyme bears similarity to a variety of redox enzyme systems, in particular those in which a cytochrome b ₅-like domain is fused to proteins carrying other redox functions.     

Effects of uranyl ions on lipid bilayer membranes

Uranyl ions (UO₂²⁺) stabilize black lipid membranes (BLM's) as inferred from the doubling of the breakdown voltage and from a considerable increase in the lifetime of the BLM's. These effects are observed also in BLM's made of mono-olein and of oxidized cholesterol. The lytic effect of lysolecithin is significantly reduced in the presence of UO₂²⁺. Uranyl ions adsorb to the interface of BLM's made of phosphatidylcholine (PC) with a dissociation constant of about 3 : 10^?6 M and thereby charge the interface of the membrane and attain almost stoichiometric binding of one molecule of uranyl ion per one molecule of PC at 1 M ionic strength and 20 μM of UO₂²⁺. The membrane conductance induced by ionophores is considerably reduced by UO₂²⁺ and it is inferred by various tests that this is due to the charging of the interface and not to changes in membrane fluidity.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H₂O₂ on cytosolic as well as mitochondrial calcium (Ca²⁺) concentrations, mitochondrial inner membrane potential (_m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H₂O₂, in the absence of extracellular Ca²⁺ ([Ca²⁺]_o) led to an increase either in cytosolic and in mitochondrial Ca²⁺ concentration. Additionally, H₂O₂ induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H₂O₂ on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H₂O₂. However, the H₂O₂-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca²⁺ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H₂O₂-evoked changes in mitochondrial Ca²⁺ concentration but not those in membrane potential and FAD state. The present results have indicated that H₂O₂ can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H₂O₂. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas. (Mol Cell Biochem 269: 165–173, 2005)     

Direct electrochemical analyses of human cytochromes <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> with a mutated heme pocket showed a good correlation between their midpoint and half wave potentials

Background  

Cytochrome b ₅ performs central roles in various biological electron transfer reactions, where difference in the redox potential of two reactant proteins provides the driving force. Redox potentials of cytochromes b ₅ span a very wide range of ~400 mV, in which surface charge and hydrophobicity around the heme moiety are proposed to have crucial roles based on previous site-directed mutagenesis analyses.     

Manipulating the Length of the <Emphasis Type="Italic">b</Emphasis> Subunit F<Subscript>1</Subscript> Binding Domain in F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP Synthase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The peripheral stalk of F₁F₀ ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F₀ to the top of the F₁ sector. The stalk serves as the stator necessary for holding F₁ against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F₁. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F₁F₀ complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F₁F₀ ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F₁F₀ complex by a normal b subunit.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Molecular modeling of cytochrome b₅ with a single cytochrome c-like thioether linkage

Bovine liver cytochrome b ₅ (cyt b ₅), with heme bound noncovalently, has been converted into a cyt c-like protein (cyt b ₅ N57C) by constructing a thioether linkage between the heme and the engineered cysteine residue. With no X-ray or NMR structure available, we herein performed a molecular modeling study of cyt b ₅ N57C. On the other hand, using amino acid sequence information for a newly discovered member of the cyt b ₅ family, domestic silkworm cyt b ₅ (DS cyt b ₅), we predicted the protein structure by homology modeling in combination with MD simulation. The modeling structure shows that both Cys57 in cyt b ₅ N57C, and Cys56, a naturally occurring cysteine in DS cyt b ₅, have suitable orientations to form a thioether bond with the heme 4-vinyl group, as the heme is in orientation A. In addition to providing structural information that was not previously obtained experimentally, these modeling studies provide insight into the formation of cyt c-like thioether linkages in cytochromes, and suggest that c-type cyt b ₅ maturation involves a b-type intermediate.     

Sugar interaction with uranium ion. Synthesis,spectroscopic and stmctural analysis of UO2-sugar complexes containing D-glucuronate moiety

Interaction between D-glucuronic acid and hydrated uranyl salts has been studied in aqueous solution and solid complexes of the type UO₂(D- glucuronate)X·2H₂0 and UO₂(D-glucuronate)₂·2H₂O, where X = CI⁻, Br⁻ or NO₃⁻, are isolated and characterized by means of FT-IR and proton-NMR spectroscopy.On comparison with the structurally identified Ca(D-glucuronate)Br·3H₂O compound, it is concluded that the UO₂²⁺ cation binds to two D- glucuronate moieties in uranylsugar complexes via O6, O5 oxygen atoms (ionized carboxyl group) of the first and O6′, 04 (non-ionized carboxyl group) of the second sugar moiety, whereas in the UO₂(D- glucuronate)₂·2H₂O salt the uranyl ion is bonded to two sugar anions through O6, O6′ oxygen atoms of the ionized carboxyl group, resulting in a six- coordination geometry around the uranium ion. The strong intermolecular hydrogen bonding network of the free acid is rearranged upon sugar metalation and the sugar moiety showed β-anomer conformation both in the free acid and in these uranylsugar complexes.