Stopped-flow Apparatus Research Articles

Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: Role of local internal viscosity in protein dynamics

Upon activation of trypsinogen four peptide segments flanked by hinge glycine residues undergo conformational changes. To test whether the degree of conformational freedom of hinge regions affects the rate of activation, we introduced amino acid side chains of different characters at one of the hinges (position 193) and studied their effects on the rate constant of the conformational change. This structural rearrangement leading to activation was triggered by a pH-jump and monitored by intrinsic fluorescence change in the stopped-flow apparatus. We found that an increase in the size of the side chain at position 193 is associated with the decrease of the reaction rate constant. To analyze the thermodynamics of the reaction, temperature dependence of the reaction rate constants was examined in a wide temperature range (5-60 degrees C) using a novel temperature-jump/stopped-flow apparatus developed in our laboratory. Our data show that the mutations do not affect the activation energy (the exponential term) of the reaction, but they significantly alter the preexponential term of the Arrhenius equation. The effect of solvent viscosity on the rate constants of the conformational change during activation of the wild type enzyme and its R193G and R193A mutants was determined and evaluated on the basis of Kramers' theory. Based on this we propose that the reaction rate of this conformational transition is regulated by the internal molecular friction, which can be specifically modulated by mutagenesis in the hinge region.

Stimuli-Responsive Double Hydrophilic Block Copolymer Micelles with Switchable Catalytic Activity

Double hydrophilic block copolymers, poly(N-isopropylacrylamide)-b-poly(N-vinylimidazole) (PNIPAM-b-PVim), were successfully prepared with good control via reversible addition−fragmentation chain transfer (RAFT) using PNIPAM-based macromolecular xanthate agents (i.e., MADIX, macromolecular design via the interchange of xanthates). This represents the first preparation of well-defined block copolymers based on PVim, which has been well-known to be able to catalyze esterolysis reactions. The imidazole-containing diblock copolymers molecularly dissolve at low temperatures in water. Above the phase transition temperatures of PNIPAM or in a proper mixture of methanol/water (cononsolvency), the PNIPAM block becomes hydrophobic and stable micelles form with a dense core consisting of a hydrophobic PNIPAM block and a polar PVim shell. The catalytic activities of PNIPAM44-b-PVim51 and PNIPAM44-b-PVim21 toward the hydrolysis of p-nitrophenyl acetate (NPA) at different temperatures or methanol/water compositions were then determined using a stopped-flow apparatus and compared to that of PVim homopolymer. The Arrhenius plot for the PVim-based diblock copolymers exhibited a pronounced upward curvature above the critical micellization temperature (cmt). Moreover, in the methanol/water mixture, the catalytic activities of PNIPAM-b-PVim diblock copolymers evolved discontinuously as a function of solvent composition and exhibited a maximum in the range of volume fraction of methanol, φmethanol, between 0.3 and 0.5, corresponding to the solvent composition range where cononsolvency-induced micellization took place. We thus observed for the first time that double hydrophilic block copolymer micelles of PNIPAM-b-PVim can serve as self-catalyzing nanoreactors. Most importantly, the catalytic activities can be well-tuned with external temperature or solvent compositions.

Effect of Mixing on the Formation of Complexes of Hyperbranched Cationic Polyelectrolytes and Anionic Surfactants

The effect of different mixing protocols on the charged nature and size distribution of the aqueous complexes of hyperbranched poly(ethylene imine) (PEI) and sodium dodecyl sulfate (SDS) was investigated by electrophoretic mobility and dynamic light scattering measurements at different pH values, polyelectrolyte concentrations, and ionic strengths. It was found that at large excess of the surfactant a colloidal dispersion of individual PEI/SDS nanoparticles forms via an extremely rapid mixing of the components by means of a stop-flow apparatus. However, the application of a less efficient mixing method under the same experimental conditions might result in large clusters of the individual PEI/SDS particles as well as in a more extended precipitation regime compared with the results of stop-flow mixing protocol. The study revealed that the larger the charge density and concentration of the PEI, the more pronounced the effect of mixing becomes. It can be concluded that an efficient way to avoid precipitation in the solutions of oppositely charged polyelectrolytes and surfactants might be provided by extending the range of kinetically stable colloidal dispersion of polyelectrolyte/surfactant nanoparticles via the application of appropriate mixing protocols.

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the beta-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to approximately 1000 s(-1), a very similar rate constant to MHC-2, but 10-15% of the complex must go through a slow isomerization (approximately 20 s(-1)) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin.ADP and provide evidence of three closely related actin-myosin.ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (approximately 20 s(-1)), and a third complex that releases ADP too slowly (approximately 6 s(-1)) to be on the main ATPase pathway. The role of these actin-myosin.ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.

Using in situ Raman monitoring as a tool for rapid optimisation and scale-up of microwave-promoted organic synthesis: esterification as an example

Microwave-promoted esterification reactions have been monitored using in situ Raman spectroscopy. Having optimised a reaction on a 23 mmol scale, it was transferred to a larger reaction vessel and scaled up to 0.26 mol, again with Raman monitoring. With conditions in hand, an automated stop-flow apparatus was used to prepare 5.7 moles of product.

A Millisecond Infrared Stopped-Flow Apparatus

In this paper, we have described a stopped-flow apparatus that is capable of measuring infrared kinetics in the amide I' region of a protein's vibrational spectrum. The dead time of this setup, determined by the reducing reaction of 2,6-Dichlorophenolindophenol by L-ascorbic acid, is between 6 to 15 ms, depending on the flow rate. Therefore, this stopped-flow IR method provides a means of measuring infrared kinetics in a time window that is not easily accessible to other mixing-based IR techniques. Using this apparatus, we have studied the alkaline transition of cytrochrome c and have found that this conformational event proceeds in a biphasic manner. The characteristic time constants of these two phases were determined to be 68 +/- 20 ms and 624 +/- 37 ms, respectively.

Micelle Formation and Inversion Kinetics of a Schizophrenic Diblock Copolymer

It is known that the zwitterionic diblock copolymer, poly(4-vinylbenzoic acid)-b-poly(N-(morpholino)ethyl methacrylate) (VBA-b-MEMA), exhibits interesting “schizophrenic” micellization behavior (see: Liu, S.; Armes, S. P. Langmuir 2003, 19, 4432−4438). The kinetics of the pH-induced formation and dissociation of VBA−core micelles, the salt-induced formation and dilution-induced dissociation of MEMA−core micelles at pH 10, and the pH-induced micellar inversion between VBA− and MEMA−core micelles in the presence of 0.8 M Na2SO4 were studied in detail using stopped-flow apparatus equipped with a light scattering detector. A pH jump from 12 to 2 in the absence of salt leads to the formation of VBA−core micelles; upon a pH jump from 2 to 12, the breakup of VBA−core micelles into unimers occurs within the dead time of the stopped-flow apparatus (∼ 2−3 ms). At pH 10, addition of Na2SO4 (> 0.6 M) induces the formation of MEMA−core micelles. Compared to the pH-induced formation and dissociation of VBA−core micell...

Conformational Transitions as Determinants of Specificity for the DNA Methyltransferase EcoRI

Changes in DNA bending and base flipping in a previously characterized specificity-enhanced M.EcoRI DNA adenine methyltransferase mutant suggest a close relationship between precatalytic conformational transitions and specificity (Allan, B. W., Garcia, R., Maegley, K., Mort, J., Wong, D., Lindstrom, W., Beechem, J. M., and Reich, N. O. (1999) J. Biol. Chem. 274, 19269-19275). The direct measurement of the kinetic rate constants for DNA bending, intercalation, and base flipping with cognate and noncognate substrates (GAATTT, GGATTC) of wild type M.EcoRI using fluorescence resonance energy transfer and 2-aminopurine fluorescence studies reveals that DNA bending precedes both intercalation and base flipping, and base flipping precedes intercalation. Destabilization of these intermediates provides a molecular basis for understanding how conformational transitions contribute to specificity. The 3500- and 23,000-fold decreases in sequence specificity for noncognate sites GAATTT and GGATTC are accounted for largely by an approximately 2500-fold increase in the reverse rate constants for intercalation and base flipping, respectively. Thus, a predominant contribution to specificity is a partitioning of enzyme intermediates away from the Michaelis complex prior to catalysis. Our results provide a basis for understanding enzyme specificity and, in particular, sequence-specific DNA modification. Because many DNA methyltransferases and DNA repair enzymes induce similar DNA distortions, these results are likely to be broadly relevant.

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.

Uncoupling the Enzymatic and Autoprocessing Activities of Helicobacter pylori γ-Glutamyltranspeptidase

Gamma-glutamyltranspeptidase (gammaGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the gamma-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported gammaGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the gamma-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian gammaGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the gamma-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions.

The Mechanism of Direct Heme Transfer from the Streptococcal Cell Surface Protein Shp to HtsA of the HtsABC Transporter

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K(d) of 120 +/- 18 microm, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6s(-1) at 25 degrees C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 +/- 0.002 s(-1). HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp.apoHtsA complex (K(d) = 48 +/- 7 microM) at a rate approximately 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k(transfer) = 43 +/- 3s(-1) versus k(-hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA (k'(hemin) approximately 80 microm(-1) s(-1), k(-hemin) = 0.0026 +/- 0.0002 s(-1)) are 50- and 10-fold greater than the corresponding rate constants for Shp (k(hemin) approximately 1.6 microM(-1) s(-1), k(-hemin) = 0.0003 s(-1)), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k(hemin) approximately 31,000 microm(-1)) is roughly 5-fold greater than that of apoShp (k(hemin) approximately 5,300 microM(-1)), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.

A Weak Fe–O Bond in the Oxygenated Complex of the Nitric-oxide Synthase of Staphylococcus aureus

Little is known about the intermediates formed during catalysis by nitric-oxide synthase (NOS). We report here the characterization by resonance Raman spectroscopy of the oxygenated complex of the NOS from Staphylococcus aureus (saNOS) as well as the kinetics of formation and decay of the complex. An oxygenated complex transiently formed after mixing reduced saNOS with oxygen and decayed to the ferric enzyme with kinetics that were dependent on the substrate L-arginine and the cofactor H(4)B. The oxygenated complex displayed a Soret absorption band centered at 430 nm. Resonance Raman spectroscopy revealed that it can be described as a ferric superoxide form (Fe(III)O(2)(-)) with a single nu(O-O) mode at 1135 cm(-1). In the presence of L-arginine, an additional nu(O-O) mode at 1123 cm(-1) was observed, indicating an increased pi back-bonding electron donation to the bound oxygen induced by the substrate. With saNOS, this is the first time that the nu(Fe-O) mode of a NOS has been observed. The low frequency of this mode, at 517 cm(-1), points to an oxygenated complex that differs from that of P450(cam). The electronic structure of the oxygenated complex and the effect of L-arginine are discussed in relation to the kinetic properties of saNOS and other NOS.

Photochemical Behavior and Na+,K+ ‐ATPase Sensitivity of Voltage‐sensitive Styrylpyridinium Fluorescent Membrane Probes

RH421 is a widely used voltage-sensitive fluorescent membrane probe. Its exposure to continuous illumination with 577 nm light from an Hg lamp leads, however, to an increase in its steady-state fluorescence level when bound to lipid membranes. The increase occurs on the second time scale at typical light intensities and was found to be due to a single-photon excited-state isomerization. Modifications to the dye structure are, therefore, necessary to increase photochemical stability and allow wider application of such dyes in kinetic studies of ion-transporting membrane proteins. The related probe ANNINE 5, which has a rigid polycyclic structure, shows no observable photochemical reaction when bound to DMPC vesicles on irradiation with 436 nm light. The voltage sensitivity of ANNINE 5 was tested with the use of Na+,K+-ATPase membrane fragments. As long as ANNINE 5 is excited on the far red edge of its visible absorption band, it shows a similar sensitivity to RH421 in detecting charge-translocating reactions triggered by ATP phosphorylation. Unfortunately the wavelengths necessary for ANNINE 5 excitation are in a region where the Hg lamps routinely used in stopped-flow apparatus have no significant lines available for excitation.

Efficient Synthesis of Sterically Stabilized pH-Responsive Microgels of Controllable Particle Diameter by Emulsion Polymerization

Emulsion polymerization of 2-vinylpyridine (2VP) in the presence of divinylbenzene (DVB) cross-linker, a cationic surfactant, and a hydrophilic macromonomer, monomethoxy-capped poly(ethylene glycol) methacrylate (PEGMA), at around neutral pH and 60 degrees C afforded near-monodisperse, sterically stabilized latexes at approximately 10% solids. Judicious selection of the synthesis parameters enabled the mean latex diameter to be varied over an unusually wide range for one-shot batch syntheses. Scanning electron microscopy studies confirmed near-monodisperse spherical morphologies, with mean weight-average particle diameters ranging from 370 to 970 nm depending on the initiator, polymeric stabilizer, and surfactant concentrations. Particle sizing studies were also conducted using disk centrifuge photosedimentometry and dynamic light scattering and gave similar data. These lightly cross-linked latexes acquired cationic microgel character at low pH, as expected. The critical pH for this latex-to-microgel transition was around pH 4.1 at 1.0 wt % DVB, which is significantly lower than the pKa of 4.92 estimated for linear P2VP homopolymer by acid titration. 1H NMR and aqueous electrophoresis studies indicated that substantial swelling occurred at low pH due to protonation of the 2VP groups, while dynamic light scattering (DLS) studies indicated volumetric swelling ratios of up to 3 orders of magnitude, depending on the initial latex diameter. Systematic variation of the degree of cross-linking led to a monotonic decrease in the pKa values of the P2VP latexes (as judged by acid titration) and also the critical swelling pH (as judged by visual inspection). This was attributed to the increasingly branched nature of the P2VP chains in their swollen microgel form. Preliminary studies of the kinetics of acid-induced swelling were also conducted using the pH jump method in conjunction with a stopped-flow apparatus. These P2VP latexes swell significantly faster than P2VP latexes described in the literature and the characteristic time scales observed in the present study are much closer to those predicted by the Tanaka equation.

A Unique ATP Hydrolysis Mechanism of Single-headed Processive Myosin, Myosin IX

Recent studies have revealed that myosin IX is a single-headed processive myosin, yet it is unclear how myosin IX can achieve the processive movement. Here we studied the mechanism of ATP hydrolysis cycle of actomyosin IXb. We found that myosin IXb has a rate-limiting ATP hydrolysis step unlike other known myosins, thus populating the prehydrolysis intermediate (M.ATP). M.ATP has a high affinity for actin, and, unlike other myosins, the dissociation of M.ATP from actin was extremely slow, thus preventing myosin from dissociating away from actin. The ADP dissociation step was 10-fold faster than the overall ATP hydrolysis cycle rate and thus not rate-limiting. We propose the following model for single-headed processive myosin. Upon the formation of the M.ATP intermediate, the tight binding of actomyosin IX at the interface is weakened. However, the head is kept in close proximity to actin due to the tethering role of loop 2/large unique insertion of myosin IX. There is enough freedom for the myosin head to find the next location of the binding site along with the actin filament before complete dissociation from the filament. After ATP hydrolysis, Pi is quickly released to form a strong actin binding form, and a power stroke takes place.

Spectral, Kinetic, and Thermodynamic Properties of Cu(I) and Cu(II) Binding by Methanobactin from Methylosinus trichosporium OB3b

To examine the potential role of methanobactin (mb) as the extracellular component of a copper acquisition system in Methylosinus trichosporium OB3b, the metal binding properties of mb were examined. Spectral (UV-visible, fluorescence, and circular dichroism), kinetic, and thermodynamic data suggested copper coordination changes at different Cu(II):mb ratios. Mb appeared to initially bind Cu(II) as a homodimer with a comparatively high copper affinity at Cu(II):mb ratios below 0.2, with a binding constant (K) greater than that of EDTA (log K = 18.8) and an approximate DeltaG degrees of -47 kcal/mol. At Cu(II):mb ratios between 0.2 and 0.45, the K dropped to (2.6 +/- 0.46) x 10(8) with a DeltaG degrees of -11.46 kcal/mol followed by another K of (1.40 +/- 0.21) x 10(6) and a DeltaG degrees of -8.38 kcal/mol at Cu(II):mb ratios of 0.45-0.85. The kinetic and spectral changes also suggested Cu(II) was initially coordinated to the 4-thiocarbonyl-5-hydroxy imidazolate (THI) and possibly Tyr, followed by reduction to Cu(I), and then coordination of Cu(I) to 4-hydroxy-5-thiocarbonyl imidazolate (HTI) resulting in the final coordination of Cu(I) by THI and HTI. The rate constant (k(obsI)) of binding of Cu(II) to THI exceeded that of the stopped flow apparatus that was used, i.e., >640 s(-)(1), whereas the coordination of copper to HTI showed a 6-8 ms lag time followed by a k(obsII) of 121 +/- 9 s(-)(1). Mb also solubilized and bound Cu(I) with a k(obsI) to THI of >640 s(-)(1), but with a slower rate constant to HTI (k(obsII) = 8.27 +/- 0.16 s(-)(1)), and appeared to initially bind Cu(I) as a monomer.

Simple and Inexpensive Computer Interface to a Durrum Stopped-Flow Apparatus Tested Using the Iron(III)–Thiocyanate Reaction

A computer has been interfaced with a Durrum model 110 stopped-flow apparatus using a LabPro unit and LoggerPro software from Vernier Software and Technology. Two separate voltage probes from Verni...

Pre-steady-state Kinetic Characterization of the DinB Homologue DNA Polymerase of Sulfolobus solfataricus

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nm, which is approximately 10-fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s(-1) for dTTP, dATP, dGTP, and dCTP (at 25 degrees C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity (K(d) approximately 600 mum), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an "induced fit" mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.

Intermolecular electron-transfer reactions in soluble methane monooxygenase: a role for hysteresis in protein function.

Electron transfer from reduced nicotinamide adenine dinucleotide (NADH) to the hydroxylase component (MMOH) of soluble methane monooxygenase (sMMO) primes its non-heme diiron centers for reaction with dioxygen to generate high-valent iron intermediates that convert methane to methanol. This intermolecular electron-transfer step is facilitated by a reductase (MMOR), which contains [2Fe-2S] and flavin adenine dinucleotide (FAD) prosthetic groups. To investigate interprotein electron transfer, chemically reduced MMOR was mixed rapidly with oxidized MMOH in a stopped-flow apparatus, and optical changes associated with reductase oxidation were recorded. The reaction proceeds via four discrete kinetic phases corresponding to the transfer of four electrons into the two dinuclear iron sites of MMOH. Pre-equilibrating the hydroxylase with sMMO auxiliary proteins MMOB or MMOD severely diminishes electron-transfer throughput from MMOR, primarily by shifting the bulk of electron transfer to the slowest pathway. The biphasic reactions for electron transfer to MMOH from several MMOR ferredoxin analogues are also inhibited by MMOB and MMOD. These results, in conjunction with the previous finding that MMOB enhances electron-transfer rates from MMOR to MMOH when preformed MMOR-MMOH-MMOB complexes are allowed to react with NADH [Gassner, G. T.; Lippard, S. J. Biochemistry 1999, 38, 12768-12785], suggest that isomerization of the initial ternary complex is required for maximal electron-transfer rates. To account for the slow electron transfer observed for the ternary precomplex in this work, a model is proposed in which conformational changes imparted to the hydroxylase by MMOR are retained throughout the catalytic cycle. Several electron-transfer schemes are discussed with emphasis on those that invoke multiple interconverting MMOH populations.

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only approximately 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is approximately 50%. More significantly, the processivity of translocation is substantially reduced by approximately 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.