Ultrafast electron transfer in the recognition of different DNA sequences by a DNA-binding protein with different dynamical conformations

Ultrafast electron transfer (ET) phenomenon in protein and protein-DNA complex is very much crucial and often leads to the regulation of various kinds of redox reactions in biological system. Although, the conformation of the protein in protein-DNA complex is concluded to play the key role in the ET process, till date very little evidences exist in the literature. λ-repressor-operator DNA interaction, particularly O(R)1 and O(R)2, is a key component of the λ-genetic switch and is a model system for understanding the chemical principles of the conformation-dependent ET reaction, governed by differential protein dynamics upon binding with different DNA target sequences. Here, we have explored the photoinduced electron transfer from the tryptophan moieties of the protein λ-repressor to two operators DNA of different sequences (O(R)1 and O(R)2) using picosecond-resolved fluorescence spectroscopy. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with O(R)1 DNA compared to O(R)2 DNA are found to have pronounced effect on the rate of ET. We have also observed the ET phenomenon from a dansyl chromophore, bound to the lysine residue, distal from the DNA-binding domain of the protein to the operator DNA with a specific excitation at 299 nm wavelength. The altered ET dynamics as a consequence of differential protein conformation upon specific DNA sequence recognition may have tremendous biological implications.     

Structural and mutational studies of the carboxylate cluster in iron-free ribonucleotide reductase R2

The R2 protein of ribonucleotide reductase features a di-iron site deeply buried in the protein interior. The apo form of the R2 protein has an unusual clustering of carboxylate side chains at the empty metal-binding site. In a previous study, it was found that the loss of the four positive charge equivalents of the diferrous site in the apo protein appeared to be compensated for by the protonation of two histidine and two carboxylate side chains. We have studied the consequences of removing and introducing charged residues on the local hydrogen-bonding pattern in the region of the carboxylate cluster of Corynebacterium ammoniagenes and Escherichia coli protein R2 using site-directed mutagenesis and X-ray crystallography. The structures of the metal-free forms of wild-type C. ammoniagenes R2 and the mutant E. coli proteins D84N, S114D, E115A, H118A, and E238A have been determined and their hydrogen bonding and protonation states have been structurally assigned as far as possible. Significant alterations to the hydrogen-bonding patterns, protonation states, and hydration is observed for all mutant E. coli apo proteins as compared to wild-type apo R2. Further structural variations are revealed by the wild-type apo C. ammoniagenes R2 structure. The protonation and hydration effects seen in the carboxylate cluster appear to be due to two major factors: conservation of the overall charge of the site and the requirement of electrostatic shielding of clustered carboxylate residues. Very short hydrogen-bonding distances between some protonated carboxylate pairs are indicative of low-barrier hydrogen bonding.     

Re-engineering redox-sensitive green fluorescent protein for improved response rate

Redox-sensitive variants of the green fluorescent protein (roGFPs) had previously been developed that allow "real-time" monitoring of the redox status of cellular compartments by fluorescence excitation ratiometry. However, the response time of these probes limits the study of certain rapid oxidative events, such as H2O2 bursts in cell signaling. The substitution of up to three positively charged amino acids adjacent to the introduced disulfide in roGFP1 (variants designated roGFP1-R1 through -R14) substantially improved the response rate. The pseudo first-order rate constants for oxidation by H2O2 and reduction by DTT and redox midpoint potentials were determined. The rate constants approximately doubled with each additional positively charged substitution, to nearly an order of magnitude total. The midpoint potentials are highly correlated with the rate increases, becoming more oxidizing with increasing numbers of positive substitutions. Crystal structures of two variants with opposite disulfide oxidation states have been determined: a 2.2 A resolution structure of oxidized "R7" containing two basic substitutions, and a 1.95 A resolution structure of reduced "R8" with one basic and one acidic substitution. Nonlinear Poisson-Boltzmann (PB) calculations are shown to accurately predict the effects of the substitutions on the rate constants. The effects of the substitutions on dimer formation, relative oxidative midpoint potentials, and oxidation and reduction rates are discussed. roGFPs are demonstrated to constitute an excellent model system for quantitative analysis of factors influencing thiol transfer reactions. roGFP1-R12 is most suitable for use in live cells, due to significantly increased reaction rate and increased pI.     

Crystallographic investigation of the role of aspartate 95 in the modulation of the redox potentials of Desulfovibrio vulgaris flavodoxin

The side chain of aspartate 95 in flavodoxin from Desulfovibrio vulgaris provides the closest negative charge to N(1) of the bound FMN in the protein. Site-directed mutagenesis was used to substitute alanine, asparagine, or glutamate for this amino acid to assess the effect of this charge on the semiquinone/hydroquinone redox potential (E(1)) of the FMN cofactor. The D95A mutation shifts the E(1) redox potential positively by 16 mV, while a negative shift of 23 mV occurs in the oxidized/semiquinone midpoint redox potential (E(2)). The crystal structures of the oxidized and semiquinone forms of this mutant are similar to the corresponding states of the wild-type protein. In contrast to the wild-type protein, a further change in structure occurs in the D95A mutant in the hydroquinone form. The side chain of Y98 flips into an energetically more favorable edge-to-face interaction with the bound FMN. Analysis of the structural changes in the D95A mutant, taking into account electrostatic interactions at the FMN binding site, suggests that the pi-pi electrostatic repulsions have only a minor contribution to the very low E(1) redox potential of the FMN cofactor when bound to apoflavodoxin. Substitution of D95 with glutamate causes only a slight perturbation of the two one-electron redox potentials of the FMN cofactor. The structure of the D95E mutant reveals a large movement of the 60-loop (residues 60-64) away from the flavin in the oxidized structure. Reduction of this mutant to the hydroquinone causes the conformation of the 60-loop to revert back to that occurring in the structures of the wild-type protein. The crystal structures of the D95E mutant imply that electrostatic repulsion between a carboxylate on the side chain at position 95 and the phenol ring of Y98 prevents rotation of the Y98 side chain to a more energetically favorable conformation as occurs in the D95A mutant. Replacement of D95 with asparagine has no effect on E(2) but causes E(1) to change by 45 mV. The D95N mutant failed to crystallize. The K(d) values of the protein FMN complex in all three oxidation-reduction states differ from those of the wild-type complexes. Molecular modeling showed that the conformational energy of the protein changes with the redox state, in qualitative agreement with the observed changes in K(d), and allowed the electrostatic interactions between the FMN and the surrounding groups on the protein to be quantified.     

Conformational changes in redox pairs of protein structures

Disulfides are conventionally viewed as structurally stabilizing elements in proteins but emerging evidence suggests two disulfide subproteomes exist. One group mediates the well known role of structural stabilization. A second redox‐active group are best known for their catalytic functions but are increasingly being recognized for their roles in regulation of protein function. Redox‐active disulfides are, by their very nature, more susceptible to reduction than structural disulfides; and conversely, the Cys pairs that form them are more susceptible to oxidation. In this study, we searched for potentially redox‐active Cys Pairs by scanning the Protein Data Bank for structures of proteins in alternate redox states. The PDB contains over 1134 unique redox pairs of proteins, many of which exhibit conformational differences between alternate redox states. Several classes of structural changes were observed, proteins that exhibit: disulfide oxidation following expulsion of metals such as zinc; major reorganisation of the polypeptide backbone in association with disulfide redox‐activity; order/disorder transitions; and changes in quaternary structure. Based on evidence gathered supporting disulfide redox activity, we propose disulfides present in alternate redox states are likely to have physiologically relevant redox activity.     

X-ray studies on crystalline complexes involving amino acids and peptides. XIX. Effects of change in chirality in the complexes of succinic acid with DL- and L-arginine

Crystals of DL-arginine hemisuccinate dihydrate (I)(monoclinic; P2(1)/c; a = 5.292, b = 16.296. c = 15.203 A; beta = 92.89 degrees; Z = 4) and L-arginine hemisuccinate hemisuccinic acid monohydrate (II) (triclinic; P1; a = 5.099; b = 10.222, c = 14.626 A; alpha = 77.31, beta = 89.46, gamma = 78.42 degrees; Z = 2) were grown under identical conditions from aqueous solutions of the components in molar proportions. The structures were solved by direct methods and refined to R = 0.068 for 2585 observed reflections in the case of (I) and R = 0.036 for 2154 observed reflections in the case of (II). Two of the three crystallographically independent arginine molecules in the complexes have conformations different from those observed so far in the crystal structures containing arginine. The succinic acid molecules and the succinate ions in the structures are centrosymmetric and planar. The crystal structure of (II) is highly pseudosymmetric. Arginine-succinate interactions in both the complexes involve specific guanidyl-carboxylate interactions. The basic elements of aggregation in both the structures are ribbons made up of alternating arginine dimers and succinate ions. However, the ribbons pack in different ways in the two structures. (II) presents an interesting case in which two ionisation states of the same molecule coexist in a crystal. The two complexes provide a good example of the effect of change in chirality on stoichiometry, conformation, aggregation, and ionisation state in the solid state.     

Temperature-dependent structural transition following X-ray-induced metal center reduction in oxidized cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in the inner membrane of mitochondria. It contains four metal redox centers, two of which, Cu_B and heme a₃, form the binuclear center (BNC), where dioxygen is reduced to water. Crystal structures of CcO in various forms have been reported, from which ligand-binding states of the BNC and conformations of the protein matrix surrounding it have been deduced to elucidate the mechanism by which the oxygen reduction chemistry is coupled to proton translocation. However, metal centers in proteins can be susceptible to X-ray-induced radiation damage, raising questions about the reliability of conclusions drawn from these studies. Here, we used microspectroscopy-coupled X-ray crystallography to interrogate how the structural integrity of bovine CcO in the fully oxidized state (O) is modulated by synchrotron radiation. Spectroscopic data showed that, upon X-ray exposure, O was converted to a hybrid O∗ state where all the four metal centers were reduced, but the protein matrix was trapped in the genuine O conformation and the ligands in the BNC remained intact. Annealing the O∗ crystal above the glass transition temperature induced relaxation of the O∗ structure to a new R∗ structure, wherein the protein matrix converted to the fully reduced R conformation with the exception of helix X, which partly remained in the O conformation because of incomplete dissociation of the ligands from the BNC. We conclude from these data that reevaluation of reported CcO structures obtained with synchrotron light sources is merited.     

Effects of redox potential and Ca2+ on the inositol 1,4,5-trisphosphate receptor L3-1 loop region: implications for receptor regulation

Inositol 1,4,5-trisphosphate receptor (IP(3)R) is a major intracellular Ca(2+) channel, modulated by many factors in the cytosolic and lumenal compartments. Compared with cytosolic control, lumenal-side regulation has been much less studied, and some of its mechanistic aspects have been controversial. Of particular interest with regard to lumenal regulation are whether it involves direct interactions between IP(3)R and the regulators, and whether it involves conformational changes of the lumenal regions of IP(3)R. To understand these lumenal-side regulation mechanisms, we studied the effects of two important lumenal regulatory factors, the redox potential and Ca(2+), on the L3-1 lumenal loop region of IP(3)R. The redox potential exerted direct and significant effects on the conformation of the loop region. By sharp contrast, Ca(2+) showed little effect on the L3-1 conformation, suggesting that the regulation of Ca(2+) is indirect or involves other receptor regions. GSH/oxidized glutathione-mediated oxidation introduced a unique intramolecular disulfide bond between Cys(34) and Cys(42). A variety of NMR experiments revealed that oxidation also induces localized helical characteristics in the Cys(34)-Cys(42) region. Dynamics studies also showed reduced motions in the region upon oxidation, consistent with the conformational changes. The results raise the interesting possibility that Cys(34) and Cys(42) may act together as a reduction sensor, and that Cys(65) may function as an oxidation sensor. Overall, our studies suggest that the redox potential and Ca(2+) can regulate IP(3)R through totally different mechanisms: Ca(2+) by the indirect effect and the redox potential by direct action causing conformational changes.     

Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex

The three-dimensional structures of the native cytochrome c(2) from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 A and in the reduced state at 1.95 A resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c(2) with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3(10)-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c(2) with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles phi and psi of approximately -140 and -130 degrees, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 A resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c(2) of the rare six-residue insertion in the helix 3(10) conformation that increases Met loop flexibility.     

Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways

Versatile peroxidases (VP), a recently described family of ligninolytic peroxidases, show a hybrid molecular architecture combining different oxidation sites connected to the heme cofactor. High-resolution crystal structures as well as homology models of VP isoenzymes from the fungus Pleurotus eryngii revealed three possibilities for long-range electron transfer for the oxidation of high redox potential aromatic compounds. The possible pathways would start either at Trp164 or His232 of isoenzyme VPL, and at His82 or Trp170 of isoenzyme VPS1. These residues are exposed, and less than 11 A apart from the heme. With the purpose of investigating their functionality, two single mutations (W164S and H232F) and one double mutation (W164S/P76H) were introduced in VPL that: (i) removed the two pathways in this isoenzyme; and (ii) incorporated the absent putative pathway. Analysis of the variants showed that Trp164 is required for oxidation of two high redox potential model substrates (veratryl alcohol and Reactive Black 5), whereas the two other pathways (starting at His232 and His82) are not involved in long-range electron transfer (LRET). None of the mutations affected Mn2+ oxidation, which would take place at the opposite side of the enzyme. Substitution of Trp164 by His also resulted in an inactive variant, indicating that an indole side-chain is required for activity. It is proposed that substrate oxidation occurs via a protein-based radical. For the first time in a ligninolytic peroxidase such an intermediate species could be detected by low-temperature electron paramagnetic resonance of H2O2-activated VP, and was found to exist at Trp164 as a neutral radical. The H2O2-activated VP was self-reduced in the absence of reducing substrates. Trp164 is also involved in this reaction, which in the W164S variant was blocked at the level of compound II. When analyzing VP crystal structures close to atomic resolution, no hydroxylation of the Trp164 Cbeta atom was observed (even after addition of several equivalents of H2O2). This is in contrast to lignin peroxidase Trp171. Analysis of the crystal structures of both peroxidases showed differences in the environment of the protein radical-forming residue that could affect its reactivity. These variations would also explain differences found for the oxidation of some high redox potential aromatic substrates.     

Structures of rat cytosolic PEPCK: insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid

The structures of the rat cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK) reported in the PEPCK-Mn2+, -Mn2+-oxaloacetic acid (OAA), -Mn2+-OAA-Mn2+-guanosine-5'-diphosphate (GDP), and -Mn2+-Mn2+-guanosine-5'-tri-phosphate (GTP) complexes provide insight into the mechanism of phosphoryl transfer and decarboxylation mediated by this enzyme. OAA is observed to bind in a number of different orientations coordinating directly to the active site metal. The Mn2+-OAA and Mn2+-OAA-Mn2+GDP structures illustrate inner-sphere coordination of OAA to the manganese ion through the displacement of two of the three water molecules coordinated to the metal in the holo-enzyme by the C3 and C4 carbonyl oxygens. In the PEPCK-Mn2+-OAA complex, an alternate bound conformation of OAA is present. In this conformation, in addition to the previous interactions, the C1 carboxylate is directly coordinated to the active site Mn2+, displacing all of the waters coordinated to the metal in the holo-enzyme. In the PEPCK-Mn2+-GTP structure, the same water molecule displaced by the C1 carboxylate of OAA is displaced by one of the gamma-phosphate oxygens of the triphosphate nucleotide. The structures are consistent with a mechanism of direct in-line phosphoryl transfer, supported by the observed stereochemistry of the reaction. In the catalytically competent binding mode, the C1 carboxylate of OAA is sandwiched between R87 and R405 in an environment that would serve to facilitate decarboxylation. In the reverse reaction, these two arginines would form the CO2 binding site. Comparison of the Mn2+-OAA-Mn2+GDP and Mn2+-Mn2+GTP structures illustrates a marked difference in the bound conformations of the nucleotide substrates in which the GTP nucleotide is bound in a high-energy state resulting from the eclipsing of all three of the phosphoryl groups along the triphosphate chain. This contrasts a previously determined structure of PEPCK in complex with a triphosphate nucleotide analogue in which the analogue mirrors the conformation of GDP as opposed to GTP. Last, the structures illustrate a correlation between conformational changes in the P-loop, the nucleotide binding site, and the active site lid that are important for catalysis.     

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.     

Synthesis,structure and catechol-oxidase activity of copper(II) complexes of 17-hydroxy-16-(N-3-oxo-prop-1-enyl)amino steroids

Copper is next to iron the most important element in the biological transport, storage and in redox reactions of dioxygen. A bioanalogous activation of dioxygen with copper complexes is used for catalytical epoxidation, allylic hydroxylation and oxidative coupling of aromatic substrates, for example. With stereochemical information in form of chiral ligands, enantioselective reactions may be possible. Another aspect of interest on copper catalyzed reactions with dioxygen is that the exact mechanism and biological function of some enzymes (especially catechol oxidase) is yet not fully clear. For studies mimicking the copper-containing catechol oxidase appropriate chiral steroid ligands with defined stereochemistry and conformation have been synthesized. The four diastereomeric 16,17-aminoalcohols of the 3-methoxy-estra-1,3,5(10)-triene series have been condensed with salicylic aldehyde and different beta-ketoenols to the chiral ligand types 1-5. These compounds with different steric and electronic properties and different arrangements of the neighboring hydroxy and nitrogen functions were reacted with copper(II) acetate to copper complexes. The structure of these complexes will be discussed. The bioanalogous oxidation of 3,5-di-tbutyl-catechol (dtbc) to the corresponding quinone was catalyzed by most of the complexes, indicating their ability to activate dioxygen. The trans configurations c and d showed an activity one magnitude higher than the cis configurations a and b. Comparing compounds with the same diastereomeric configuration, the main influence was that of the peripheral R(1-3) substituents at the beta-ketoenaminic group which are useful for the fine-tuning of the properties of the copper atoms like redox potential and Lewis acidity.     

Hydrogen sulfide concentration in the milieu of the hydrated alanine dipeptide determines its polyproline II-beta propensity: Main chain contribution to the energetic origin of the formation of amyloid

In view of the observation that the concentration of hydrogen sulfide in brains with Alzheimer’s disease (AD) is lower than that in normal brains and in line with our previous studies indicating that additional content in the aqueous environment (milieu) of a peptide can change its local energetic preference from a polyproline II (P) to a β conformation (and therefore its tendency to form the β-chain structures that lead to the amyloid plaques associated with the disease), we have studied the effect of H₂S concentration on such propensity in a simple model peptide, the alanine dipeptide (ADP). The two concentration states are represented by ADP(H₂O)₁₈(H₂S) and ADP(H₂O)₁₈(H₂S)₂. Ab initio calculations of these structures show that the lowest energy of the former is a β conformation while that of the latter is a P, mirroring the observed AD results and strengthening our proposal that amyloid diseases are better viewed in the context of a protein milieu-folding paradigm.     

Time-resolved infrared detection of the proton and protein dynamics during photosynthetic oxygen evolution

Photosynthetic oxygen evolution by plants and cyanobacteria is performed by water oxidation at the Mn(4)CaO(5) cluster in photosystem II. The reaction is known to proceed via a light-driven cycle of five intermediates called S(i) states (i = 0-4). However, the detailed reaction processes during the intermediate transitions remain unresolved. In this study, we have directly detected the proton and protein dynamics during the oxygen-evolving reactions using time-resolved infrared spectroscopy. The time courses of the absorption changes at 1400 and 2500 cm(-1), which represent the reactions and/or interaction changes of carboxylate groups and the changes in proton polarizability of strong hydrogen bonds, respectively, were monitored upon flash illumination. The results provided experimental evidence that during the S(3) → S(0) transition, drastic proton rearrangement, most likely reflecting the release of a proton from the catalytic site, takes place to form a transient state before the oxidation of the Mn(4)CaO(5) cluster that leads to O(2) formation. Early proton movement was also detected during the S(2) → S(3) transition. These observations reveal the common mechanism in which proton release facilitates the transfer of an electron from the Mn(4)CaO(5) cluster in the S(2) and S(3) states that already accumulate oxidizing equivalents. In addition, relatively slow rearrangement of carboxylate groups was detected in the S(0) → S(1) transition, which could contribute to the stabilization of the S(1) state. This study demonstrates that time-resolved infrared detection is a powerful method for elucidating the detailed molecular mechanism of photosynthetic oxygen evolution by pursuing the reactions of substrate and amino acid residues during the S-state transitions.     

Di-iron—carboxylate proteins

Di-iron centers bridged by carboxylate residues and oxide/hydroxide groups have so far been seen in four classes of proteins involved in dioxygen chemistry or phosphoryl transfer reactions. The dinuclear iron centers in these proteins are coordinated by histidines and additional carboxylate ligands. Recent structural data on some of these enzymes, combined with spectroscopic and kinetic data, can now serve as a base for detailed mechanistic suggestions. The di-iron sites in the major class of hydroxylase-oxidase enzymes, which contains ribonucleotide reductase and methane monooxygenase, show significant flexibility in the geometry of their coordination of three or more carboxylate groups. This flexibility, combined with a relatively low coordination number, and a buried environment suitable for reactive oxygen chemistry, explains their efficient harnessing of the oxidation power of molecular oxygen.     

Oxidized and reduced Azotobacter vinelandii ferredoxin I at 1.4 A resolution: conformational change of surface residues without significant change in the [3Fe-4S]+/0 cluster.

The refined structure of reduced Azotobacter vinelandii 7Fe ferredoxin FdI at 100 K and 1.4 A resolution is reported, permitting comparison of [3Fe-4S]+ and [3Fe-4S]0 clusters in the same protein at near atomic resolution. The reduced state of the [3Fe-4S]0 cluster is established by single-crystal EPR following data collection. Redundant structures are refined to establish the reproducibility and accuracy of the results for both oxidation states. The structure of the [4Fe-4S]2+ cluster in four independently determined FdI structures is the same within the range of derived standard uncertainties, providing an internal control on the experimental methods and the refinement results. The structures of the [3Fe-4S]+ and [3Fe-4S]0 clusters are also the same within experimental error, indicating that the protein may be enforcing an entatic state upon this cluster, facilitating electron-transfer reactions. The structure of the FdI [3Fe-4S]0 cluster allows direct comparison with the structure of a well-characterized [Fe3S4]0 synthetic analogue compound. The [3Fe-4S]0 cluster displays significant distortions with respect to the [Fe3S4]0 analogue, further suggesting that the observed [3Fe-4S]+/0 geometry in FdI may represent an entatic state. Comparison of oxidized and reduced FdI reveals conformational changes at the protein surface in response to reduction of the [3Fe-4S]+/0 cluster. The carboxyl group of Asp15 rotates approximately 90 degrees, Lys84, a residue hydrogen bonded to Asp15, adopts a single conformation, and additional H2O molecules become ordered. These structural changes imply a mechanism for H+ transfer to the [3Fe-4S]0 cluster in agreement with electrochemical and spectroscopic results.     

Oxidation and nuclear localization of thioredoxin-1 in sparse cell cultures

Reactive oxygen species (ROS) were once viewed only as mediators of toxicity, but it is now recognized that they also contribute to redox signaling through oxidation of specific cysteine thiols on regulatory proteins. Cells in sparse cultures have increased ROS relative to confluent cultures, but it is not known whether protein redox states are affected under these conditions. The purpose of the present study was to determine whether culture conditions affect the redox state of thioredoxin-1 (Trx1), the protein responsible for reducing most oxidized proteins in the cytoplasm and nucleus. The results showed that Trx1 was more oxidized in sparse HeLa cell cultures than in confluent cells. The glutathione pool was also more oxidized, demonstrating that both of the major cellular redox regulating systems were affected by culture density. In addition, the total amount of Trx1 protein was lower and the subcellular distribution of Trx1 was different in sparse cells. Trx1 in sparse cultures was predominantly nuclear whereas it was predominantly cytoplasmic in confluent cultures. This localization pattern was not unique to HeLa cells as it was also observed in A549, Cos-1 and HEK293 cells. These findings demonstrate that Trx1 is subject to changes in expression, redox state and subcellular localization with changing culture density, indicating that the redox environments of the cytoplasm and the nucleus are distinct and have different requirements under different culture conditions.     

Reactions of glutathione and glutathione radicals with benzoquinones.

The reactions of glutathione (GSH) and glutathione radicals with a series of methyl-substituted 1,4-benzoquinones and 1,4-benzoquinone have been studied. It was found that by mixing excess benzoquinone with glutathione at pH above 6.5, the products formed were complex and unstable. All of the other experiments were carried out at pH 6.0, where the main product was stable for several hours. Stopped-flow analysis allowed the measurement of the rates of the rapid reactions between GSH and the quinones, and the products were monitored by High Performance Liquid Chromatography (HPLC). The rates of the reactions vary by five orders of magnitude and must be influenced by steric factors as well as changes in the redox states. It was observed that simple hydroquinones were not formed when the different benzoquinones were mixed with excess GSH and suggests that the initial reaction is addition/reduction rather than electron transfer. In the presence of excess quinone, the hydroquinone of the glutathione conjugate is oxidized back to its quinone. The rates of the reaction were measured. By using the technique of pulse radiolysis, it was possible to measure the reduction of the quinones by GSSG.- and the oxidation of hydroquinones by GS(.). It is proposed that the appearance of GSSG in reactions of quinones with glutathione could be due to oxidation of the hydroquinone by oxygen and the subsequent superoxide or H2O2 promoting the oxidation of GSH to GSSG.     

Kinetic differences at low temperatures between R and T state carbon monoxide-carp hemoglobin.

We use the low-temperature recombination kinetics of carbon monoxide with carp hemoglobin to determine that the R and T states of hemoglobin exhibit different low-temperature geminate recombination kinetics. The peak of the fitted Gaussian activation energy spectrum is at 1.5 kcal/mol for R state and 1.8 kcal/mol for T state. The distribution in activation energies is fit well by the Agmon-Hopfield linear strain model. The T state is fit with a stronger elastic constant than R, and has a larger displacement of the protein conformation coordinate than does the R state, indicating that the T state does have a significantly greater rigidity and also stores more strain energy in its conformational states than does R hemoglobin. The pre-exponential in the activation energy spectrum is only a factor of two greater in the R than the T state and the low-temperature activation energy spectrum does not correctly predict the difference in the on rates for R and T states at 300 degrees K, indicating that processes removed from the binding site are important in cooperativity.