Stopped-flow Kinetic Analysis Research Articles

ArsH from the Cyanobacterium Synechocystis sp. PCC 6803 Is an Efficient NADPH-Dependent Quinone Reductase

The cyanobacterium Synechocystis sp. PCC 6803 possesses an arsenic resistance operon that encodes, among others, an ArsH protein. ArsH is a flavin mononucleotide (FMN)-containing protein of unknown function and a member of the family of NADPH-dependent FMN reductases. The nature of its final electron acceptor and the role of ArsH in the resistance to arsenic remained to be clarified. Here we have expressed and purified Synechocystis ArsH and conducted an intensive biochemical study. We present kinetic evidence supporting a quinone reductase activity for ArsH, with a preference for quinones with hydrophobic substituents. By using steady-state activity measurements, as well as stopped-flow and laser-flash photolysis kinetic analyses, it has been possible to establish the mechanism of the process and estimate the values of the kinetic constants. Although the enzyme is able to stabilize the anionic semiquinone form of the FMN, reduction of quinones involves the hydroquinone form of the flavin cofactor, and the enzymatic reaction occurs through a ping-pong-type mechanism. ArsH is able to catalyze one-electron reactions (oxygen and cytocrome c reduction), involving the FMN semiquinone form, but with lower efficiency. In addition, arsH mutants are sensitive to the oxidizing agent menadione, suggesting that ArsH plays a role in the response to oxidative stress caused by arsenite.

NBD-Labeled Phospholipid Accelerates Apolipoprotein C-II Amyloid Fibril Formation but Is Not Incorporated into Mature Fibrils

Human apolipoprotein (apo) C-II is one of several lipid-binding proteins that self-assemble into fibrils and accumulate in disease-related amyloid deposits. A general characteristic of these amyloid deposits is the presence of lipids, known to modulate individual steps in amyloid fibril formation. ApoC-II fibril formation is activated by submicellar phospholipids but inhibited by micellar lipids. We examined the mechanism for the activation by submicellar lipids using the fluorescently labeled, short-chain phospholipid 1-dodecyl-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-2-hydroxyglycero-3-phosphocholine (NBD-lyso-12-PC). Addition of submicellar NBD-lyso-12-PC increased the rate of fibril formation by apoC-II approximately 2-fold. Stopped flow kinetic analysis using fluorescence detection and low, non-fibril-forming concentrations of apoC-II indicated NBD-lyso-12-PC binds rapidly, on the millisecond time scale, followed by the slower formation of discrete apoC-II tetramers. Sedimentation velocity analysis showed NBD-lyso-12-PC binds to both apoC-II monomers and tetramers at approximately five sites per monomer with an average dissociation constant of approximately 10 μM. Mature apoC-II fibrils formed in the presence of NBD-lyso-12-PC were devoid of lipid, indicating a purely catalytic role for submicellar lipids in the activation of apoC-II fibril formation. These studies demonstrate the catalytic potential of small amphiphilic molecules in controlling protein folding and fibril assembly pathways.

Identification of DypB from Rhodococcus jostii RHA1 as a Lignin Peroxidase

Rhodococcus jostii RHA1, a polychlorinated biphenyl-degrading soil bacterium whose genome has been sequenced, shows lignin degrading activity in two recently developed spectrophotometric assays. Bioinformatic analysis reveals two unannotated peroxidase genes present in the genome of R. jostii RHA1 with sequence similarity to open reading frames in other lignin-degrading microbes. They are members of the Dyp peroxidase family and were annotated as DypA and DypB, on the basis of bioinformatic analysis. Assay of gene deletion mutants using a colorimetric lignin degradation assay reveals that a ΔdypB mutant shows greatly reduced lignin degradation activity, consistent with a role in lignin breakdown. Recombinant DypB protein shows activity in the colorimetric assay and shows Michaelis-Menten kinetic behavior using Kraft lignin as a substrate. DypB is activated by Mn(2+) by 5-23-fold using a range of assay substrates, and breakdown of wheat straw lignocellulose by recombinant DypB is observed over 24-48 h in the presence of 1 mM MnCl(2). Incubation of recombinant DypB with a β-aryl ether lignin model compound shows time-dependent turnover, giving vanillin as a product, indicating that C(α)-C(β) bond cleavage has taken place. This reaction is inhibited by addition of diaphorase, consistent with a radical mechanism for C-C bond cleavage. Stopped-flow kinetic analysis of the DypB-catalyzed reaction shows reaction between the intermediate compound I (397 nm) and either Mn(II) (k(obs) = 2.35 s(-1)) or the β-aryl ether (k(obs) = 3.10 s(-1)), in the latter case also showing a transient at 417 nm, consistent with a compound II intermediate. These results indicate that DypB has a significant role in lignin degradation in R. jostii RHA1, is able to oxidize both polymeric lignin and a lignin model compound, and appears to have both Mn(II) and lignin oxidation sites. This is the first detailed characterization of a recombinant bacterial lignin peroxidase.

Stopped-flow kinetic analysis of the interaction of cyclo[8]pyrrole with anions.

The on and off rates corresponding to the binding of two test anions (acetate, AcO(-), and dihydrogen phosphate, H(2)PO(4)(-), studied as their tetrabutylammonium salts) to diprotonated cyclo[8]pyrrole have been determined in CH(3)CN using stopped-flow analyses carried out at various temperatures. For dihydrogen phosphate, this afforded the activation enthalpies and entropies associated with both off and on processes. The different dynamic behavior seen for these test anions underscores the utility of kinetic analyses as a possible new tool for the advanced characterization of anion receptors.

Flexible Recognition of the tRNA G18 Methylation Target Site by TrmH Methyltransferase through First Binding and Induced Fit Processes

Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes methyl transfer from S-adenosyl-l-methionine to a conserved G18 in tRNA. We investigated the recognition mechanism of Thermus thermophilus TrmH for its guanosine target. Thirteen yeast tRNA(Phe) mutant transcripts were prepared in which the modification site and/or other nucleotides in the D-loop were substituted by dG, inosine, or other nucleotides. We then conducted methyl transfer kinetic studies, gel shift assays, and inhibition experiments using these tRNA variants. Sites of methylation were confirmed with RNA sequencing or primer extension. Although the G18G19 sequence is not essential for methylation by TrmH, disruption of G18G19 severely reduces the efficiency of methyl transfer. There is strict recognition of guanosine by TrmH, in that methylation occurs at the adjacent G19 when the G18 is replaced by dG or adenosine. The fact that TrmH methylates guanosine in D-loops from 4 to 12 nucleotides in length suggests that selection of the position of guanosine within the D-loop is relatively flexible. Our studies also demonstrate that the oxygen 6 atom of the guanine base is a positive determinant for TrmH recognition. The recognition process of TrmH for substrate is inducible and product-inhibited, in that tRNAs containing Gm18 are excluded by TrmH. In contrast, substitution of G18 with dG18 results in the formation of a more stable TrmH-tRNA complex. To address the mechanism, we performed the stopped-flow pre-steady state kinetic analysis. The result clearly showed that the binding of TrmH to tRNA is composed of at least three steps, the first bi-molecular binding and the subsequent two uni-molecular induced-fit processes.

Growth Mechanisms in Nanocrystalline Lead Sulfide by Stopped-Flow Kinetic Analysis

The growth mechanisms of thiolate-capped lead sulfide nanocrystals have been elucidated using stopped-flow UV−visible absorption spectroscopy. Spectra were recorded over the range of 0−45 °C at 48 ms intervals during the first 144 s of reaction between Pb(NO3)2 and Na2S in an aqueous solution containing thioglycerol and dithioglycerol capping ligands at pH 11. The spectra were deconvoluted by fitting to three Gaussian curves; the lowest-energy of these corresponded to the first excitonic transition and was used to approximate the average particle radius using a linear relationship between the band gap and the particle diameter for spherical PbS clusters. Kinetic models for oriented attachment (OA) and Ostwald ripening (OR) growth mechanisms were used to interpret the evolution of particle radius with time. Least-squares fitting analysis revealed that OR was the single dominant mechanism of growth in the low temperature range (0−25 °C), while at higher temperatures (30−45 °C) the mechanism was time dependent: within the first 20 s, growth was predominantly by OR; OA was favored thereafter. Parametric optimization showed that OR was controlled by the dissolution kinetics at the particle−matrix interface. The enthalpy (ΔH‡) and entropy (ΔS‡) of activation for the OA process were determined by Eyring treatment to be 12 ± 3 and −160 ± 10 J K−1 mol−1, respectively; the resulting Gibbs free energy of activation (ΔG‡) was 62 ± 6 kJ mol−1. The very early stages of nanocrystal growth (<0.5 s) were dominated by nucleation processes, and the kinetic data could be fit to neither OR nor OA models.

Identification And Characterization Of Cardiac Troponin I From The Trout Heart

Trout cardiac myofibrils are ∼10-fold more sensitive to Ca+2 than those from mammalian hearts when measured at the same temperature. It has been demonstrated that trout cardiac troponin C (ScTnC) has 2.3 fold the Ca2+ affinity of human cTnC and is responsible for a 2-fold increase in cardiac myofibril Ca+2 sensitivity. The contributions of trout cardiac troponin I (ScTnI) to the Ca+2 sensitivity of the trout heart is currently unknown. The cDNA for ScTnI has been cloned using RACE-PCR. Sequencing results indicate that ScTnI is 59% and 56% identical to human cTnI and human skeletal troponin I, respectively, at the amino acid level. Interestingly, ScTnI lacks the ∼30-residue N-terminal sequence present in mammalian cTnI that contains two protein kinase A (PKA) target residues at positions 23 and 24. ScTnI has been expressed and the influence of it on the Ca2+ activation of human cardiac troponin is currently being characterized using steady state Ca2+ binding assays and stopped flow kinetic analysis. This work is supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Foundation for Innovation (CFI) to TEG.

Structural Analysis of Substrate and Effector Binding in Mycobacterium tuberculosis d-3-Phosphoglycerate Dehydrogenase

The crystal structure of Mycobacterium tuberculosis d-3-phosphoglycerate dehydrogenase has been solved with bound effector, l-serine, and substrate, hydroxypyruvic acid phosphate, at resolutions of 2.7 and 2.4 A, respectively. The subunits display the same extreme asymmetry as seen in the apo-structure and provide insight into the mode of serine binding and closure of the active site. Mutagenesis studies confirm the identity of the main residues involved in serine binding and suggest that the poly glycine stretch in the loop that contains the locus for the 160 degrees rotation that leads to subunit asymmetry may have a larger role in folding than in catalysis. The lack of electron density for the cofactor, NADH, in any of the crystals examined led us to study binding by stopped flow kinetic analysis. The kinetic data suggest that productive NADH binding, that would support catalytic turnover, is dependent on the presence of substrate. This observation, along with the binding of substrate in the active site, but in an unproductive conformation, suggests a possible mechanism where initial binding of substrate leads to enhanced interaction with cofactor accompanied by a rearrangement of catalytically critical residue side chains. Furthermore, comparison to the structure of a truncated form of human d-3-phosphoglycerate dehydrogenase with cofactor and a substrate analog, provides insight into the conformational changes that occur during catalysis.

Partition of mismatch recognition between MutSα and MutSβ

DNA mismatch repair plays an important role in genome maintenance. MutS α (MSH2–MSH6) and MutS β (MSH2–MSH3) are two major mismatch repair proteins in mammalian cells that preferentially recognize base‐base and small (1‐2 nucleotides) insertion/deletion (ID) mispairs, respectively. Normally, cells maintain a MutSα:MutSβ ratio at ~ 10:1. Overexpressing MSH3 alters the MutSα:MutSβ ratio, leading to a mutator phenotype. To determine the molecular basis by which mispairs are differentially recognized by MutSα and MutS β, purified proteins were tested for interactions, independently or competitively, with a G‐T or an ID heteroduplex using gel shift and stopped‐flow kineticanalyses. Although MutS α bound both the G‐T and ID substrates, the affinity of the protein to the G‐T duplex is ~ 1.5‐fold higher than that to the ID duplex. Surprisingly, MutS β could also interact with the G‐T substrate, but the binding affinity was only ~ 1/3 of the activity that the protein had for the ID substrate. These relative affinities measured in these equilibrium binding and competition experiments were confirmed by direct measurement of association and dissociation kinetic constants by stopped‐flow spectroscopy. The order of binding activities among these proteins and DNA substrates is MutS β ‐ID &gt; MutSα‐G‐T &gt; MutSα‐ID &gt; MutS β ‐G‐T. Our findings therefore explain why cells maintain a high MutSα:MutSβ ratio.

Regulation of translation elongation by guanine nucleotide exchange factor, eEF1Bα

Translation elongation requires the GTP-dependent activity of eEF1A. eEF1Bα functions as the catalytic subunit of the guanine nucleotide exchange factor complex of eEF1A to maintain the GTP-bound form and allow ternary complex (eEF1A-GTP-aminoacyl tRNA) formation. Structural analysis shows that domain II of eEF1A forms a hydrophobic pocket that binds eEF1Bα and is proposed to overlap the aa-tRNA binding site. Since F163 of eEF1Bα is nested in this pocket, it may stabilize eEF1A binding and channeling of aa-tRNA to eEF1A. The F163A mutant exhibits growth and translation defects. We hypothesized that W130A eEF1Bα mutation increases flexibility to compensate for the F163A defect causing intragenic suppression. W130A/F163A double mutant partially restores F163A defects in vivo. Stopped-flow kinetic analysis demonstrates that F163A eEF1Bα decreases GDP exchange 30 fold and has a higher Kd for eEF1A of 26 μM compared to 4 μM for WT. The W130A mutant increases koff of the GDP:eEF1A complex 2 fold. The W130A/F163A mutant partially suppresses F163A exchange activity but does not compensate for its eEF1A binding defect. This data suggests that intragenic suppression is due to the rate at which eEF1Bα accelerates GDP release. Further studies will utilize these eEF1Bα mutations to understand the interplay between eEF1A:eEF1Bα complex formation and the formation of the aa-tRNA:eEF1A:GTP ternary complex. Supported by NIH.

Structural Basis of the Transition-state Stabilization in Antibody-catalyzed Hydrolysis

The catalytic antibody 6D9, which was raised against a transition-state analogue (TSA), catalyzes the hydrolysis of a non-bioactive chloramphenicol monoester to generate chloramphenicol. It has been shown that 6D9 utilizes the binding affinity in the catalysis; the differential affinity of the TSA relative to the substrate is equal to the rate enhancement. To reveal the recognition mechanism of 6D9 for the TSA and the substrate, we performed NMR analysis of the Fv fragment of 6D9 (6D9-Fv), together with site-directed mutagenesis and stopped-flow kinetic analyses. Among six 6D9-Fv mutants, Y58(H)A and W100i(H)A displayed significant reductions in their affinities to the TSA, while their substrate-binding affinities were identical with that of the wild-type 6D9-Fv. The stopped-flow kinetic studies revealed that the TSA binding to 6D9-Fv occurred by an induced-fit mechanism. In contrast, no induced-fit type of TSA-binding mechanism was observed for Y58(H)A and W100i(H)A. From NMR experiments, we identified the residues with chemical shifts that were perturbed by the ligand-binding. The residues affected by the TSA binding were located on the TSA-binding site determined by the X-ray study, and on the regions far from the binding site. On the other hand, the residues affected by the substrate binding were localized on the TSA-binding site. As for W100i(H)A, no residue other than those in the binding site was affected by the ligand binding. On the basis of these results and the crystal structure, we concluded that the TSA binding induced a conformational change involving the formation of aromatic–aromatic interactions and a hydrogen bond. These interactions can account for the differential affinity for the TSA relative to the substrate. W100i(H) probably plays an important role in inducing the conformational changes. The present NMR studies have enabled us to visualize the concept of transition-state stabilization in enzymatic catalysis, in which the transition-state contacts are better than those of the substrate.

Millisecond Association Kinetics of K+ with Triazacryptand-Based K+ Indicators Measured by Fluorescence Correlation Spectroscopy

We recently introduced a water-soluble, long-wavelength K(+)-sensing indicator, TAC-Red, consisting of a triazacryptand K(+)-selective ionophore coupled to a xanthylium chromophore (Nat. Methods 2005, 2, 825-827). Stopped-flow kinetic analysis indicated that in response to changes in K(+) concentration TAC-Red fluorescence enhancement occurs in milliseconds or less. Here, we use fluorescence correlation spectroscopy to quantify the binding kinetics of K(+) with TAC-Red and a new, longer-wavelength sensor, TAC-Crimson. Autocorrelation functions, G(tau), were similar at 0 and high (150 mM) K(+) concentrations, with the appearance of a prominent kinetic process with a correlation time in the millisecond range for K(+) concentrations between approximately 20 and 60 mM. Control experiments with increased illumination volume and solution viscosity indicated that the millisecond component represented K(+)/TAC-Red association. K(+)-dependent G(tau) data, modeled using a global regression to a binding/diffusion model, gave association and dissociation rate constants of 0.0020 +/- 0.0003 mM(-1) ms(-1) and 0.12 +/- 0.02 ms(-1), respectively, for TAC-Red. Similar results were obtained for TAC-Crimson. The rapid K(+) binding kinetics with triazacryptand-based sensors support their utility for measuring changes in K(+) concentrations during rapid neural signaling and ion channel gating.

Binding and Transport of Metal Ions at the Dimer Interface of the Escherichia coli Metal Transporter YiiP

YiiP is a representative member of the cation diffusion facilitator (CDF) family, a class of ubiquitous metal transporters that play an essential role in metal homeostasis. Recently, a pair of Zn2+/Cd2+-selective binding sites has been localized to two highly conserved aspartyl residues (Asp157), each in a 2-fold-symmetry-related transmembrane segment 5 (TM5) of a YiiP homodimer. Here we report the functional and structural interactions between Asp157 and yet another highly conserved Asp49 in the TM2. Calorimetric binding analysis indicated that Asp49 and Asp157 contribute to a common Cd2+ binding site in each subunit. Copper phenanthroline oxidation of YiiP(D49C), YiiP(D157C), and YiiP(D49C/D157C) yielded inter- and intra-subunit cross-links among Cys49 and Cys157, consistent with the spatial proximity of two (Asp49-Asp157) sites at the dimer interface. Hg2+ binding to YiiP(D49C) or YiiP(D49C/D157C) also yielded a Cys49-Hg2+-Cys49 biscysteinate complex across the dimer interface, further establishing the interfacial location of a (Asp49-Asp157)2 bimetal binding center. Two bound Cd2+ ions were found transported cooperatively with a sigmoidal dependence on the Cd2+ concentration (n = 1.4). The binding affinity, transport cooperativity, and rate were modestly reduced by either a D49C or D157C mutation, but greatly diminished when all the bidentate aspartate O-ligands in (Asp49-Asp157)2 were replaced by the monodentate cysteine S-ligands. The functional significance of these findings is discussed based on the unique coordination chemistry of aspartyl residues and a model for the translocation pathway of metal ions at the YiiP dimer interface.

Role of the His57−Glu214 Ionic Couple Located in the Active Site ofMycobacterium tuberculosisFprA,

Mycobacterium tuberculosis FprA is a NADPH-ferredoxin reductase, functionally and structurally similar to the mammalian adrenodoxin reductase. It is presumably involved in supplying electrons to one or more of the pathogen's cytochrome P450s through reduced ferredoxins. It has been proposed on the basis of crystallographic data (Bossi, R. T., et al. (2002) Biochemistry 41, 8807-8818) that the highly conserved His57 and Glu214 whose side chains are H-bonded are involved in catalysis. Both residues were individually changed to nonionizable amino acyl residues through site-directed mutagenesis. Steady-state kinetics showed that the role of Glu214 in catalysis is negligible. On the contrary, the substitutions of His57 markedly impaired the catalytic efficiency of FprA for ferredoxin in the physiological reaction. Furthemore, they decreased the k(cat)/K(m) value for NADPH in the ferricyanide reduction. Rapid-reaction (stopped-flow) kinetic analysis of the isolated reductive half-reaction of wild-type and His57Gln forms of FprA with NADPH and NADH allowed a detailed description of the mechanism of enzyme-bound FAD reduction, with the identification of the intermediates involved. The His57Gln mutation caused a 6-fold decrease in the rate of hydride transfer from either NADPH or NADH to the enzyme-bound FAD cofactor. The 3D structure of FprA-H57Q, obtained at 1.8 A resolution, explains the inefficient hydride transfer of the mutant in terms of a suboptimal geometry of the nicotinamide-isoalloxazine interaction in the active site. These data demonstrate the role of His57 in the correct binding of NADPH to FprA for the subsequent steps of the catalytic cycle to proceed at a high rate.

Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides: EVIDENCE FOR A ONE-STEP BINDING MECHANISM

Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5' -terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m(7)GpppG, the rate constant of association, k(on), was dependent on ionic strength, decreasing progressively up to 350 mm KCl, but the rate constant of dissociation, k(off), was independent of ionic strength. Phosphorylation of eIF4E decreased k(on) by 2.1-2.3-fold at 50-100 mm KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mm. Contrary to published evidence, eIF4E phosphorylation had no effect on k(off). Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single- to double-exponential as the eIF4E concentration was increased. However, measuring k(off) for dissociation of a pre-formed eIF4E.m(7)GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in k(off). This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.

Biochemical and mutational analysis of the eukaryotic translation elongation guanine nucleotide exchange factor eEF1Bα

Translation elongation requires the GTP-dependent activity of the eEF1 complex, which in S. cerevisiae has three subunits: eEF1A, eEF1Bα, and eEF1Bγ. To sustain efficient protein synthesis, eEF1Bα functions as the guanine nucleotide exchange factor (GEF) of eEF1A to maintain the active GTP-bound form. Stopped-flow kinetic analysis demonstrates that eEF1Bα stimulates nucleotide exchange 700-fold. Structures predict K205 of eEF1Bα promotes GEF activity by disruption of the Mg2+ binding site and the lethality of a K205A mutant is not due to a structural defect. The K205A mutant drastically reduces exchange activity, but the K205R mutation is functional in vivo with nearly wild type GDP dissociation rates. Mg2+ does not affect the exchange activity of K205R mutant, however K205A does not respond to the inhibitory effect of Mg2+ on exchange activity. This data demonstrates the role of K205 and Mg2+ in the nucleotide exchange on eEF1A. F163 of eEF1Bα binds to a hydrophobic pocket of eEF1A proposed to overlap the aa-tRNA binding site. We hypothesized that the hydrogen bond between W130 and E192 of eEF1Bα allows proper conformation for binding stability to eEF1A and exchange activity, however, loss of this bond constraint allows compensation for the F163A defect. In vivo suppression analysis confirms this model. These mutations allow us to dissect the contributions of eEF1A:eEF1Bα binding on nucleotide exchange and aa-tRNA binding, thereby providing us with specific insight on G-protein regulation.

Recognizing and Defining True Ras Binding Domains I: Biochemical Analysis

Common domain databases contain sequence motifs which belong to the ubiquitin fold family and are called Ras binding (RB) and Ras association (RalGDS/AF6 Ras associating) (RA) domains. The name implies that they bind to Ras (or Ras-like) GTP-binding proteins, and a few of them have been documented to qualify as true Ras effectors, defined as binding only to the activated GTP-bound form of Ras. Here we have expressed a large number of these domains and investigated their interaction with Ras, Rap and M-Ras. While their (albeit weak) sequence homology suggest that the domains adopt a common fold, not all of them bind to Ras proteins, irrespective of whether they are called RB or RA domains. We used fluorescence spectroscopy and isothermal titration calorimetry to show that the binding affinities vary over a large range, and are usually specific for either Ras or Rap. Moreover, the specificity is dictated by a set of key residues in the interface. Stopped-flow kinetic analysis showed that the association rate constants determine the different affinities of effector binding, while the dissociation rate constants are in a similar range. Manual sequence analysis allowed us to define positively charged sequence epitopes in certain secondary structure elements of the ubiquitin fold (beta1, beta2 and alpha1) which are located at similar positions and comprise the hot spots of the binding interface. These residues are important to qualify an RA/RB domain as a true candidate Ras or Rap effector.

Kinetic studies of iron deposition catalyzed by recombinant human liver heavy, and light ferritins and Azotobacter vinelandii bacterioferritin using O 2 and H 2O 2 as oxidants

The discrepancy between predicted and measured H 2O 2 formation during iron deposition with recombinant heavy human liver ferritin (rHF) was attributed to reaction with the iron protein complex [Biochemistry 40 (2001) 10832–10838]. This proposal was examined by stopped-flow kinetic studies and analysis for H 2O 2 production using (1) rHF, and Azotobacter vinelandii bacterial ferritin (AvBF), each containing 24 identical subunits with ferroxidase centers; (2) site-altered rHF mutants with functional and dysfunctional ferroxidase centers; and (3) recombinant human liver light ferritin (rLF), containing no ferroxidase center. For rHF, nearly identical pseudo-first-order rate constants of 0.18 s −1 at pH 7.5 were measured for Fe 2+ oxidation by both O 2 and H 2O 2, but for rLF, the rate with O 2 was 200-fold slower than that for H 2O 2 ( k=0.22 s −1). A Fe 2+/O 2 stoichiometry near 2.4 was measured for rHF and its site altered forms, suggesting formation of H 2O 2. Direct measurements revealed no H 2O 2 free in solution 0.5–10 min after all Fe 2+ was oxidized at pH 6.5 or 7.5. These results are consistent with initial H 2O 2 formation, which rapidly reacts in a secondary reaction with unidentified solution components. Using measured rate constants for rHF, simulations showed that steady-state H 2O 2 concentrations peaked at 14 μM at ∼600 ms and decreased to zero at 10–30 s. rLF did not produce measurable H 2O 2 but apparently conducted the secondary reaction with H 2O 2. Fe 2+/O 2 values of 4.0 were measured for AvBF. Stopped-flow measurements with AvBF showed that both H 2O 2 and O 2 react at the same rate ( k=0.34 s −1), that is faster than the reactions with rHF. Simulations suggest that AvBF reduces O 2 directly to H 2O without intermediate H 2O 2 formation.

Thermodynamic and Kinetic Analysis of the Nitrosyl, Carbonyl, and Dioxy Heme Complexes of Neuronal Nitric-oxide Synthase: THE ROLES OF SUBSTRATE AND TETRAHYDROBIOPTERIN IN OXYGEN ACTIVATION

Mammalian NO synthases catalyze the monooxygenation of L-arginine (L-Arg) to N-hydroxyarginine (NOHA) and the subsequent monooxygenation of this to NO and citrulline. Both steps proceed via formation of an oxyferrous heme complex and may ultimately lead to a ferrous NO complex, from which NO must be released. Electrochemical reduction of NO-bound neuronal nitricoxide synthase (nNOS) oxygenase domain was used to form the ferrous heme NO complex, which was found to be stable only in the presence of low NO concentrations, due to catalytic degradation of NO at the nNOS heme site. The reduction potential for the heme-NO complex was approximately -140 mV, which shifted to 0 mV in the presence of either L-Arg or NOHA. This indicates that the complex is stabilized by 14 kJ mol(-1) in the presence of substrate, consistent with a strong H-bonding interaction between NO and the guanidino group. Neither substrate influenced the reduction potential of the ferrous heme CO complex, however. Both L-Arg and NOHA appear to interact with bound NO in a similar way, indicating that both bind as guanidinium ions. The dissociation constant for NO bound to ferrous heme in the presence of l-Arg was determined electrochemically to be 0.17 nM, and the rate of dissociation was estimated to be 10(-4) s(-1), which is much slower than the rate of catalysis. Stopped-flow kinetic analysis of oxyferrous formation and decay showed that both l-Arg and NOHA also stabilize the ferrous heme dioxy complex, resulting in a 100-fold decrease in its rate of decay. Electron transfer from the active-site cofactor tetrahydrobiopterin (H4B) has been proposed to trigger the monoxygenation process. Consistent with this, substitution by the analogue/inhibitor 4-amino-H4B stabilized the oxyferrous complex by a further two orders of magnitude. H4B is required, therefore, to break down both the oxyferrousand ferrous nitrosyl complexes of nNOS during catalysis. The energetics of these processes necessitates an electron donor/acceptor operating within a specific reduction potential range, defining the role of H4B.

Autocatalytic formation of green heme: evidence for H2O2-dependent formation of a covalent methionine-heme linkage in ascorbate peroxidase.

The mammalian heme peroxidases are distinguished from their plant and fungal counterparts by the fact that the heme group is covalently bound to the protein through ester links from glutamate and aspartate residues to the heme 1- and 5-methyl groups and, in the case of myeloperoxidase, through an additional sulfonium link from the Cbeta of the 2-vinyl group to a methionine residue. To duplicate the sulfonium link in myeloperoxidase and to obtain information on its mechanism of formation, we have engineered a methionine residue close to the 2-vinyl group in recombinant pea cytosolic ascorbate peroxidase (rpAPX) by replacement of Ser160 by Met (S160M variant). The S160M variant is isolated from Escherichia coli as apo-protein. Reconstitution of apo-S160M with exogenous heme gives a red protein (S160M(R)) which has UV-visible (lambda(max)/nm = 407, 511, 633) and steady-state kinetic (kcat = 156 +/- 7 s(-1), KM = 102 +/- 15 microM) properties that are analogous to those of rpAPX. The reaction of S160M(R) with H2O2 gives a green protein (S160M(G)). Electronic spectroscopy, mass spectrometry, and HPLC analyses are consistent with the formation of a covalent linkage between the methionine residue and the heme vinyl group in S160M(G). Single-wavelength and photodiode array stopped-flow kinetic analyses identify a transient Compound I species as a reaction intermediate. The results provide the first direct evidence that covalent heme linkage formation occurs as an H2O2-dependent process that involves Compound I formation. A mechanism that is consistent with the data is presented.