Stopped-flow Spectrophotometry Research Articles

A Stopped-flow Study on the Kinetics and Mechanism of CO2 Uptake by the cis-[Cr(1,10-phenanthroline)2(OH2)2]3+ Complex Ion

The kinetics of the reaction between gaseous CO2 and the cis-[Cr(phen)2(OH2)2]3+ ion leading to the formation of the carbonato complex ion, have been studied over the pH and temperature ranges: 3 < pH < 6 and 5 < T < 25 °C, respectively, at a constant ionic strength of 1 m (NaClO4). Investigations were carried out using the stopped-flow spectrophotometry technique in the UV–Vis range: 340–700 nm. The major reactant species in the pH range studied was cis-[Cr(phen)2(OH)(OH2)]2+ ion, which underwent reaction with CO2 to form cis-[Cr(phen)2(OH2)(HCO3)]2+ ion. Subsequently, slower ring closure of the latter species to form the bidentate carbonato chelate was observed. The possible mechanism has been discussed and the activation parameters ΔH† and ΔS† were also determined for the reaction studied.

Kinetic and structural characterization of human manganese superoxide dismutase containing 3-fluorotyrosines

Incorporation of 3-fluorotyrosine and site-specific mutagenesis have been used with stopped-flow spectrophotometry and pulse radiolysis to investigate the catalytic properties of human manganese superoxide dismutase (MnSOD). All of the nine tyrosine residues in each of the four subunits of the homotetramer of human MnSOD were replaced with 3-fluorotyrosine. Previous studies showed that the crystal structures of the unfluorinated and fluorinated human MnSOD are nearly superimposable with the root-mean-square deviation for 198 α-carbon atoms at 0.3 Å. However, the catalytic activity k cat/ K m of the fluorinated MnSOD at 30 μM −1 s −1 was less than unfluorinated wild type at 800 μM −1 s −1. Comparison of the values of k cat/ K m for fluorinated and unfluorinated wild-type andY34F MnSOD showed that this decrease for the fluorinated enzyme was in significant part due to 3-fluorotyrosine residues distant (>7 Å) from the active-site metal, not to 3-fluorotyrosine at position 34 close (∼5 Å) to the metal. Although many rate constants for the catalysis are decreased by this fluorination, the rate of dissociation of the product-inhibited complex appears unchanged by the presence of fluorinated tyrosines. These results suggest that Tyr34 is not a proton donor in the release of the product-inhibited complex, which involves protonation of a peroxo complex of the metal with release of hydrogen peroxide.

Global Effects of the Energetics of Coenzyme Binding: NADPH Controls the Protein Interaction Properties of Human Cytochrome P450 Reductase

The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'-ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K(d)), the enthalpy (DeltaH(B)) and entropy (TDeltaS(B)) of binding, and the heat capacity change (DeltaC(p)). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K(d2)(')(,5)(')(-ADP) = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (DeltaC(p)) values for 2',5'-ADP (-210 cal mol(-)(1) K(-)(1)), NADP(+) (-230 cal mol(-)(1) K(-)(1)), and H(4)NADP (-220 cal mol(-)(1) K(-)(1)) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TDeltaS(B) = -13400 +/- 200 cal mol(-)(1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TDeltaS(B) = -11200 +/- 300 cal mol(-)(1), 35 degrees C) indicate that the number of accessible conformational substates of the protein increases upon 2',5'-ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S., and Roberts, G. C. K. (2002) Biochemistry 41, 4626-4637]. A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional domain motion impinges directly on the molecular recognition of cytochrome c by CPR. Single-turnover reduction of cytochrome c by CPR (k(max) = 15 s(-)(1), K(d) = 37 microM) is critically coupled to coenzyme binding through ligand-induced motions that enable the FMN-binding domain to overcome a kinetically unproductive conformation. This is remarkable since the FMN-binding domain is not directly involved in coenzyme binding, the NADP(H) binding site being fully contained in the FAD-binding domain. Sequential rapid mixing measurements indicate that harnessing of coenzyme binding energy to the formation of a kinetically productive CPR-cytochrome c complex is a highly synchronized event. The inferred half-time for the decay of this productive conformation (tau(50)) is 330 +/- 70 ms only. Previously proposed structural and kinetic models are discussed in light of these findings.

Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide

The reaction of Euphorbia characias latex peroxidase (ELP) with hydrogen peroxide as the sole substrate was studied by conventional and stopped-flow spectrophotometry. The reaction mechanism occurs via three distinct pathways. In the first (pathway I), ELP shows catalase-like activity: H2O2 oxidizes the native enzyme to compound I and subsequently acts as a reducing substrate, again converting compound I to the resting ferric enzyme. In the presence of an excess of hydrogen peroxide, compound I is still formed and further reacts in two other pathways. In pathway II, compound I initiates a series of cyclic reactions leading to the formation of compound II and compound III, and then returns to the native resting state. In pathway III, the enzyme is inactivated and compound I is converted into a bleached inactive species; this reaction proceeds faster in samples illuminated with bright white light, demonstrating that at least one of the intermediates is photosensitive. Calcium ions decrease the rate of pathway I and accelerate the rate of pathways II and III. Moreover, in the presence of calcium the inactive stable verdohemochrome P670 species accumulates. Thus, Ca2+ ions seem to be the key for all catalytic pathways of Euphorbia peroxidase.

Kinetics and mechanism of the reactions of Pd(ii) complexes with azoles and diazines. Crystal structure of [Pd(bpma)(H2O)](ClO4)2·2H2O

Substitution reactions of the complexes [Pd(bpma)(H2O)]2+, [Pd(bpma)Cl]+, [Pd(dien)(H2O)]2+ and [Pd(dien)Cl]+, where bpma = bis(2-pyridylmethyl)amine and dien = diethylentriamine or 1,5-diamino-3-azapentane, with some nitrogen-donor ligands such as triazole, pyrazole, pyrimidine, pyrazine and pyridazine, were studied in an aqueous 0.10 M NaClO4 at pH 2.8 using variable-temperature and -pressure stopped-flow spectrophotometry. The second-order rate constants indicate that the Pd(II) complexes of bpma, viz. [Pd(bpma)(H2O)]2+ and [Pd(bpma)Cl]+, are more reactive than the complexes of dien, viz. [Pd(dien)(H2O)]2+ and [Pd(dien)Cl]+. Also, the aqua complexes, [Pd(bpma)(H2O)]2+ and [Pd(dien)(H2O)]2+, are much more reactive than the corresponding chloro complexes. The most reactive nucleophile of the five-membered rings is triazole and for the six-membered rings the most reactive one is pyridazine. Activation parameters were determined for all reactions and the negative entropies and volumes of activation (Delta S++, Delta V++) support an associative ligand substitution mechanism. The crystal structure of [Pd(bpma)(H2O)](ClO4)2.2H2O was determined by X-ray diffraction. Crystals are monoclinic with the space group P2(1)/c. The coordination geometry of [Pd(bpma)(H2O)]2+ is distorted square-planar. The Pd-N (central) bond distance, 1.958(5) A, is shorter than the other two Pd-N distances, 2.007(5) and 2.009(5) A. The Pd-O distance is 2.043(5) A.

Oxidation of resin and fatty acids by ozone: Kinetics and toxicity study

The ozonation of resin and fatty acids (RFAs) found in pulp mill effluents was investigated using rapid-scan stopped-flow spectrophotometry. RFAs oxidation (i.e., degradation) efficiency increased with increasing the amount of used ozone and temperature. The degradation process with respect to the acid was found to follow first-order kinetics. The ozonation of RFAs was modeled as an overall second-order reaction for both reactants. The apparent overall second-order rate constants were calculated based on the pseudo first-order rate constants obtained from the kinetic data fitting for the acid degradation. The apparent overall second-order rate constant was affected by pH and temperature. At 20 degrees C and when pH increased from 8 to 11, the apparent overall second-order rate constant increased almost by a factor of 5 (from 3.9 x10(3) to 1.8 x10(4)M(-1)s(-1)) for 9 mgL(-1) resin acid and a factor of 4 (from 9.6 x10(3) to 3.9 x10(4)M(-1)s(-1)) for 8 mgL(-1) fatty acid. At pH 8 and as temperature increased from 10 to 20 degrees C, the apparent overall second-order rate constant increased almost by a factor of 5 (from 8.2 x10(2) to 3.9 x10(3)M(-1)s(-1)) for 9 mgL(-1) resin acid and a factor of 3 (from 3.5 x10(3) to 9.6 x10(3)M(-1)s(-1)) for 8 mgL(-1) fatty acid. Microtox bioassay tests were completed to evaluate the toxicity of RFAs samples before and after ozonation. For the resin acid, there was an increase in toxicity as a result of ozonation. Meanwhile, toxicity of fatty acid samples decreased as a result of ozonation.

Inhibition of the Fenton reaction by nitrogen monoxide

The toxicity of iron is believed to originate from the Fenton reaction which produces the hydroxyl radical and/or oxoiron2+. The effect of nitrogen monoxide on the kinetics of the reaction of iron(II) bound to citrate, ethylenediamine-N,N'-diacetate (edda), ethylenediamine-N,N,N',N'-tetraacetate (edta), (N-hydroxyethyl)amine-N,N',N'-triacetate (hedta), and nitrilotriacetate (nta) with hydrogen peroxide was studied by stopped-flow spectrophotometry. Nitrogen monoxide inhibits the Fenton reaction to a large extent. For instance, hydrogen peroxide oxidizes iron(II) citrate with a rate constant of 5.8x10(3) M(-1) s(-1), but in the presence of nitrogen monoxide, the rate constant is 2.9x10(2) M(-1) s(-1) . Similar to hydrogen peroxide, the reaction of tert-butyl hydroperoxide with iron(II) complexes is also efficiently inhibited by nitrogen monoxide. Generally, nitrogen monoxide binds rapidly to a coordination site of iron(II) occupied by water. The rate of oxidation is influenced by the rate of dissociation of the nitrogen monoxide from iron(II).

Alkenecis-Dihydroxylation by [(Me3tacn)(CF3CO2)RuVIO2]ClO4(Me3tacn = 1,4,7-Trimethyl-1,4,7-triazacyclononane): Structural Characterization of [3 + 2] Cycloadducts and Kinetic Studies

cis-Dioxoruthenium(VI) complex [(Me(3)tacn)(CF(3)CO(2))Ru(VI)O(2)]ClO(4) (1, Me(3)tacn = 1,4,7-trimethyl-1,4,7-triazacyclononane) reacted with alkenes in aqueous tert-butyl alcohol to afford cis-1,2-diols in excellent yields under ambient conditions. When the reactions of 1 with alkenes were conducted in acetonitrile, oxidative C=C cleavage reaction prevailed giving carbonyl products in >90% yields without any cis-diol formation. The alkene cis-dihydroxylation and C=C cleavage reactions proceed via the formation of a [3 + 2] cycloadduct between 1 and alkenes, analogous to the related reactions with alkynes [Che et al. J. Am. Chem. Soc. 2000, 122, 11380]. With cyclooctene and trans-beta-methylstyrene as substrates, the Ru(III) cycloadducts (4a) and (4b) [formula; see text] were isolated and structurally characterized by X-ray crystal analyses. The kinetics of the reactions of 1 with a series of p-substituted styrenes has been studied in acetonitrile by stopped-flow spectrophotometry. The second-order rate constants varied by 14-fold despite an overall span of 1.3 V for the one-electron oxidation potentials of alkenes. Secondary kinetic isotope effect (KIE) was observed for the oxidation of beta-d(2)-styrene (k(H)/k(D) = 0.83 +/- 0.04) and alpha-deuteriostyrene (k(H)/k(D) = 0.96 +/- 0.03), which, together with the stereoselectivity of cis-alkene oxidation by 1, is in favor of a concerted mechanism.

Pore selectivity analysis of an aquaglyceroporin by stopped-flow spectrophotometry on bacterial cell suspensions

Background information. Transport of water and small neutral solutes across plasma membranes is facilitated by AQP (aquaporin) and aquaglyceroporin channels, which belong to the MIP (major intrinsic protein) family. So far, more than 800 MIP proteins have been identified on the basis of sequence homology, but only less than 10% of them have been functionally characterized. In most studies, the channel properties of MIP proteins have been determined by using Xenopus oocyte swelling assays or stopped-flow spectrophotometry on proteoliposomes. As both methods sometimes present disadvantages, we developed an alternative method for analysing MIP function.Results. The kinetics of plasmolysis or deplasmolysis of Escherichia coli cells in suspension, in response to osmotic challenges, was analysed by stopped-flow spectrophotometry. Cytoplasmic volume variations were monitored either by GFP (green fluorescent protein) fluorescence quenching or by 90 degrees scattered light. The single exponential response to up-shocks in the impermeant solute mannitol was strongly accelerated when the cells expressed the native E. coli AQP AqpZ (rate constant 37.24 versus 3.05 s(-1) for control cells). The responses to hyperosmotic shocks realized with glycerol were biphasic. First, a light-scattering increase corresponded to cell plasmolysis. Secondly, deplasmolysis occurred when glycerol entered into the cell. Both phases were accelerated when the aquaglyceroporin GlpF was present in cell membranes. We concluded that the behaviour of MIP-expressing bacteria in the stopped-flow system was qualitatively identical with that reported for MIP-expressing oocytes or MIP-containing proteoliposomes. We then used this system to analyse the effects of mutations in the pore constriction of Gla(Llac), the aquaglyceroporin from Lactococcus lactis. In the present study, we show that Gla(Llac) loses its ability to transport glycerol but retains its ability to transport water when Val(223) was replaced by a histidine, the residue at the equivalent position in strict AQPs.Conclusions. These results show that stopped-flow spectrophotometry performed on E. coli cell suspensions is a useful experimental system to analyse the selectivity of wild-type or mutant MIP proteins and that a bifunctional aquaglyceroporin switches to an AQP by a single amino acid mutation in the pore constriction.

Identification of 33 Rice Aquaporin Genes and Analysis of Their Expression and Function

Plant aquaporins form a large protein family including plasma membrane-type (PIPs) and tonoplast-type aquaporins (TIPs), and facilitate osmotic water transport across membranes as a key physiological function. We identified 33 genes for aquaporins in the genome sequence of rice (Oryza sativa L. cv. Nipponbare). We investigated their expression levels in leaf blades, roots and anthers of rice (cv. Akitakomachi) using semi-quantitative reverse transcription-PCR (RT-PCR). At both early tillering (21 d after germination) and panicle formation (56 d) stages, six genes, including OsPIP2;4 and OsPIP2;5, were expressed predominantly in roots, while 14 genes, including OsPIP2;7 and OsTIP1;2, were found in leaf blades. Eight genes, such as OsPIP1;1 and OsTIP4;1, were evenly expressed in leaf blades, roots and anthers. Analysis by stopped-flow spectrophotometry revealed high water channel activity when OsPIP2;4 or OsPIP2;5 were expressed in yeast but not when OsPIP1;1 or OsPIP1;2 were expressed. Furthermore, the mRNA levels of OsPIP2;4 and OsPIP2;5 showed a clear diurnal fluctuation in roots; they showed a peak 3 h after the onset of light and dropped to a minimum 3 h after the onset of darkness. The mRNA levels of 10 genes including OsPIP2;4 and OsPIP2;5 markedly decreased in roots during chilling treatment and recovered after warming. The changes in mRNA levels during and after the chilling treatment were comparable with that of the bleeding sap volume. These results suggested the relationship between the root water uptake and mRNA levels of several aquaporins with high water channel activity, such as OsPIP2;4 and OsPIP2;5.

Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl- d-glucamine dithiocarbamate

Iron(II)–dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)–dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N -(dithiocarboxy)sarcosine (dtcs) and N -methyl - d -glucamine dithiocarbamate (mgd) are 56 and −25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)–dtcs and iron(II)–mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)–dtcs and –mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6–3.2) × 10 4 and (0.7–1.1) × 10 5 M −1 s −1, respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)–dtcs and iron(II)–mgd by hydrogen peroxide is significantly slower (ca. 10–15 M −1 s −1). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)–dtcs and iron(III)–mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M −1 s −1, respectively.

μ-1,2-Peroxo Diferric Complex Formation in Horse Spleen Ferritin. A Mixed H/L-Subunit Heteropolymer

Previous kinetics studies with homopolymer ferritins (bullfrog M-chain, human H-chain and Escherichia coli bacterial ferritins) have established that a mu-1,2-peroxo diferric intermediate is formed during Fe(II) oxidation by O2 at the ferroxidase site of the protein. The present study was undertaken to determine whether such an intermediate is formed also during iron oxidation in horse spleen ferritin (HoSF), a naturally occurring heteropolymer ferritin of H and L-subunits (approximately 3.3 H-chains/HoSF), and to assess its role in the formation of the mineral core. Multi-wavelength stopped-flow spectrophotometry of the oxidative deposition of iron in HoSF demonstrated that a transient peroxo complex (lambda(max) approximately 650 nm) is produced in this protein as for other ferritins. The peroxo complex in HoSF is formed about fourfold slower than in human H-chain (HuHF) and decays more slowly (approximately threefold) as well, at an iron level of two Fe(II)/H-chain. However, as found for HuHF, a second intermediate is formed in HoSF as a decay product of the peroxo complex. Only one-third of the expected peroxo complex forms at the ferroxidase centers of HoSF when two Fe(II)/H-subunits are added to the protein, dropping to only approximately 14% when 20 Fe(II)/H-chain are added, indicating a declining role of the peroxo complex in iron deposition. In contrast to HuHF, HoSF does not enzymatically regenerate the observable peroxo complex. The kinetics of mineralization in HoSF are modeled satisfactorily by a mechanism in which the ferroxidase site rapidly produces an incipient core from a single turnover of iron, upon which subsequent Fe(II) is oxidized autocatalytically to build the Fe(O)OH(s) mineral core. This model supports a role for the L-chain in iron mineralization and helps to explain the widespread occurrence of heteropolymer ferritins in tissues of vertebrates.

Spectral and kinetic studies on the interaction between oxyhaemoglobin and peroxynitrite: a comparison with hydrogen peroxide

Interaction between oxyhaemoglobin and peroxynitrite was studied using stopped-flow rapid-scan spectrophotometry. The influence of pH, peroxynitrite concentration and temperature on the pseudo-first-order rate constants was studied and the activation energy calculated. The kinetic curve for the oxyhaemoglobin-peroxynitrite reaction showed that a fast reaction occurred in the initial seconds, followed by a slow process of decrease in absorbance. The biphasic reaction kinetics of oxyhaemoglobin with peroxynitrite or hydrogen peroxide demonstrated the existence of an intermediary species. For the first time a rapid-scan stopped-flow spectrophotometry study is presented, yielding spectral and kinetic data of the reaction.

Probing the role of tightly bound phosphoenolpyruvate in Escherichia coli 3-deoxy- d- manno-octulosonate 8-phosphate synthase catalysis using quantitative time-resolved electrospray ionization mass spectrometry in the millisecond time range

Escherichia coli 3-deoxy- d- manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) and d-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. The enzyme is often isolated with varying amounts of tightly bound PEP substrate. To better understand the role of tightly bound PEP in E. coli KDO8P synthase catalysis, a combination of transient kinetic methodologies including rapid chemical quench and mass spectrometry techniques such as time-resolved electrospray ionization mass spectrometry (ESI-TOF MS) were used to study the enzyme purified both in the PEP-bound state and in the unbound state. Pre-steady state burst and single-turnover experiments using radiolabeled [1- 14C] and [ 32P]A5P revealed significant kinetic differences between these enzyme preparations. The active sites concentrations for the bound and unbound states of the enzyme were almost the same (∼100%) and the product release for both states of the enzyme was rate limiting. However, the rate constant of product formation for the PEP-bound enzyme (125 s −1) was higher than that of the unbound enzyme (46 s −1). This was further confirmed by single-turnover experiments using radiolabeled [ 32P]A5P. Interestingly, when PEP was removed from the PEP-bound enzyme and external PEP was added before the kinetic experiments, both the pre-steady state burst and the single-turnover kinetic parameters were similar to those of the enzyme purified in the unbound state. The rate constants of product formation were determined as 44 s −1 (burst experiment) and 48 s −1 (single-turnover experiment). The reaction kinetics of the E. coli KDO8P synthase was also followed by time-resolved ESI mass spectrometry. To validate the suitability of this technique for conducting enzyme kinetics, the standard reaction of p-nitrophenyl acetate hydrolysis by chymotrypsin was analyzed by stopped-flow and time-resolved ESI-TOF MS. The rate constant of p-nitrophenol formation followed by stopped-flow spectrophotometry matched perfectly the rate constant of acetyl–chymotrypsin intermediate formation followed by time-resolved ESI-TOF MS (0.1 s −1). The catalytic properties of the PEP-bound and unbound states of the E. coli KDO8P synthase were then studied on a millisecond time scale. The changes in the intensity of E•PEP, E•KDO8P, and E•intermediate complexes as a function of time were quantified and the reaction kinetics were modeled using KinTekSim simulation software. An analysis of the reaction kinetics established the kinetic competence of the intermediate based upon the rate constants for substrate decay and product formation. The ability of time-resolved ESI-TOF MS to detect and monitor the kinetics for the reaction intermediate constitutes a significant advantage over the traditional rapid chemical quench technique. For all three states of the enzyme (PEP-bound, unbound, and PEP removed from the PEP-bound state) the rate constants obtained by time-resolved ESI-TOF MS matched the pre-steady state rates determined by rapid chemical quench. A comparison of reaction time courses for each state of the enzyme revealed that, in the case of PEP-bound enzyme, the enzymatic reaction reached completion faster than that for the unbound state. In summary, these studies led to the conclusion that bound PEP has an important role in catalysis, maintaining the enzyme in a conformational state optimal for catalytic activity, and established the kinetic competence of the reaction intermediate. This technique has broad applicability for the kinetic analysis of any enzyme system where the substrates, products, or intermediates are eluding the common detection techniques or as a method alternative to the widely used radioactivity assays.

Reaction of Ferric Cytochrome P450cam with Peracids: KINETIC CHARACTERIZATION OF INTERMEDIATES ON THE REACTION PATHWAY

Reactions of substrate-free ferric cytochrome P450cam with peracids to generate Fe=O intermediates have previously been investigated with contradictory results. Using stopped-flow spectrophotometry, the reaction with m-chloroperoxybenzoic acid demonstrated an Fe(IV)=O + porphyrin pi-cation radical (Cpd I) (Egawa, T., Shimada, H., and Ishimura, Y. (1994) Biochem. Biophys. Res. Commun. 201, 1464-1469). By contrast, with peracetic acid, Fe(IV)=O plus a tyrosyl radical were observed by freeze-quench Mossbauer and EPR spectroscopy (Schunemann, V., Jung, C., Trautwein, A. X., Mandon, D., and Weiss, R. (2000) FEBS Lett. 479, 149-154). Our detailed kinetic studies have resolved these contradictory results. At pH >7, a significant fraction of Cpd I is formed transiently, whereas at low pH only a species with a Soret band at 406 nm, presumably Fe(IV)=O + tyrosyl radical, is observed. Evidence for formation of an acylperoxo complex en route to Cpd I was obtained. Because of rapid heme destruction, steps subsequent to formation of the highly oxidized forms could not be fully characterized. Heme destruction was avoided by including peroxidase substrates (e.g. guaiacol), which were oxidized to characteristic peroxidase products as the Fe(III)-P450 was regenerated. Addition of ascorbate to either of the high valent species also reforms the Fe(III) state with only a small loss of heme absorbance. These results indicate that typical peroxidase chemistry occurs with P450cam and offer an explanation for the contrasting results reported earlier. The delineation of improved conditions (pH, temperature, choice of peracid) for generating highly oxidized species with P450cam should be valuable for their further characterization.

Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography

Metallo-beta-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 10(2)-10(4)-fold drops in k(cat) values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pK(a) values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pK(a) for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 A resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 A compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.

The Inner Mitochondrial Membrane Has Aquaporin-8 Water Channels and Is Highly Permeable to Water

Mitochondria are remarkably plastic organelles constantly changing their shape to fulfil their various functional activities. Although the osmotic movement of water into and out of the mitochondrion is central for its morphology and activity, the molecular mechanisms and the pathways for water transport across the inner mitochondrial membrane (IMM), the main barrier for molecules moving into and out of the organelle, are completely unknown. Here, we show the presence of a member of the aquaporin family of water channels, AQP8, and demonstrate the strikingly high water permeability (Pf) characterizing the rat liver IMM. Immunoblotting, electron microscopy, and biophysical studies show that the largest mitochondria feature the highest AQP8 expression and IMM Pf. AQP8 was also found in the mitochondria of other organs, whereas no other known aquaporins were seen. The osmotic water transport of liver IMM was partially inhibited by the aquaporin blocker Hg2+, while the related activation energy remained low, suggesting the presence of a Hg2+-insensitive facilitated pathway in addition to AQP8. It is suggested that AQP8-mediated water transport may be particularly important for rapid expansions of mitochondrial volume such as those occurring during active oxidative phosphorylation and those following apoptotic signals.

Synthesis of [Ni(η 2-CH 2C 6H 4R-4){PPh(CH 2CH 2PPh 2) 2}] + (R = H, Me or MeO) and protonation reactions with HCl

The complexes [Ni(η 2-CH 2C 6H 4R-4)(triphos)]BPh 4 {R = H, Me or MeO; triphos = PhP(CH 2CH 2PPh 2) 2} have been prepared and characterised by spectroscopy and X-ray crystallography. In all cases the coordination geometry of the nickel is best described as square-planar with an η 2-benzyl ligand occupying one of the positions. The orientation of the η 2-benzyl ligand is dictated by the steric restrictions imposed by the phenyl groups on the triphos ligand, so that the phenyl group on the unique secondary phosphorus and the aromatic group of the benzyl ligand (which are trans to one another) are oriented in the same direction. [Ni(η 2-CH 2C 6H 4R-4)(triphos)] + react with an excess of anhydrous HCl in MeCN to form [NiCl(triphos)] + (characterised as the [BPh 4] − salt by X-ray crystallography) and the corresponding substituted toluene. The kinetics of the reaction of all [Ni(η 2-CH 2C 6H 4R-4)(triphos)] + and HCl in the presence of Cl − have been determined using stopped-flow spectrophotometry. All reactions exhibit a first-order dependence on the concentration of complex and a first-order dependence on the ratio [HCl]/[Cl −]. Varying the 4-R-substituent on the benzyl ligand shows that electron-withdrawing substituents facilitate the rate of the reaction. It is proposed that the mechanism involves initial rapid protonation at the nickel to form [NiH(η 2-CH 2C 6H 4R-4)(triphos)] 2+, followed by intramolecular proton migration from nickel to carbon to yield the products.

Effect of Added Nucleophilic Species on the Rate of Primary Amino Acid Nitrosation

The rate of primary amino acid nitrosation, both with and without the addition of nucleophilic species, has been studied using stopped-flow spectrophotometry. The rate of nitrosation in the presence of strong nucleophilic species such as thiocyanate and thiourea was shown to be much faster than nitrosation without the addition of a nucleophile. Rate constants were determined at 25 degrees C for reaction of the amino acids alanine, glycine, and valine with five common nitrosating agents. For the nitrosating agents nitrosyl chloride, nitrosyl bromide, and dinitrogen trioxide the rate of reaction was observed to approach the predicted encounter-controlled limit. However, for nitrosyl thiocyanate and S-nitrosothiourea nitrosation was found to be reaction-controlled. In the reaction-controlled regime, rate constants were found to increase with increasing electrophilic strength of the nitrosating agent, as measured by the parameter En, with a slope indicative of a product-like transition state. Activation energies were also measured, being around 10-30 kJ mol-1 for encounter-controlled rate constants, and 30-50 kJ mol-1 for reaction-controlled rate constants. Our results are discussed in the context of in vivo amino acid nitrosation, where it is proposed that the rate of nitrosation may be considerably greater than currently thought, due to the presence of nucleophilic species.

Electron transfer reactions of tris(polypyridine)cobalt(iii) complexes, [Co(N–N)3]3+, with verdazyl radicals in acetonitrile solution

The kinetics and mechanisms of the reactions of 3-(4-X)-phenyl-1,5-diphenyl-verdazyl radicals where X = Cl, H, CH3 and CH3O with [Co(N-N)3]3+, N-N = 2,2'-bipyridyl (bpy), 1,10-phenanthroline (phen) and 4,7-dimethyl-1,10-phenanthroline (4,7-Me2phen), have been investigated in acetonitrile at 25 degrees C and ionic strength 0.05 mol dm(-3)(nC4H9)4NPF6 using stopped flow spectrophotometry. In all cases, transfer of one electron from the radical takes place resulting in the production of a Co(II) species and a verdazylium cation. The electron transfer occurs by an outer-sphere mechanism and the reactions appear to be consistent with Marcus theory. The self-exchange rate constants for the verdazyl-verdazylium cation have been estimated and are of the order of 3.4(+/-1.9) x 10(7) dm(3) mol(-1) s(-1). This rate constant is consistent with the fact that the reactions of [Ru(bpy)3]3+ with verdazyl radicals are too rapid to be investigated by stopped flow spectrophotometry.