On the steric course of the adenosylcobalamin-dependent 2-methyleneglutarate mutase reaction in Clostridium barkeri

The enzymatically active enantiomer of 3-methylitaconate in Clostridium barkeri has (R)-configuration. This was checked by fermentation of the racemate and reisolation of the (S)-enantiomer. In addition (R)-3-methylitaconate was synthesized by enzymatic isomerisation of 2,3-dimethylmaleate which was protonated at the Si-face. 2-Methylene[2-2H1]glutarate was synthesized via (R)-3-methyl[3-2H1]itaconate by brief incubation of 2,3-dimethylmaleate with a cell-free extract of Clostridium barkeri in 2H2O. The predominantly monodeuterated compound was oxidized to (S)-[2-2H1]succinate as analysed by circular dichroism. The results demonstrate that 2-methyleneglutarate mutase catalyses the reversible migration of an acryloyl residue from the alpha-carbon to the beta-carbon of propionate with inversion of configuration at the alpha-carbon.     

Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes

Polymorphonuclear leukocytes contain an oxidase system that can be activated to produce superoxide radicals and hydrogen peroxide. A nonmitochondrial b cytochrome, functioning in the generation of these oxygen species, has been purified to apparent homogeneity from human polymorphonuclear phagocytes. After solubilization of the cytochrome with Triton X-100, the cell extract was subsequently chromatographed on Blue Sepharose and Sephacryl S-300. The final preparation was maximally purified 170-fold with a specific content of 5.33 +/- 2.03 nmol mg-1 of protein (mean +/- S.D.; n = 7) and a yield of 21 +/- 13% (n = 5). The apparent molecular mass of the nondenatured cytochrome was estimated by gel filtration to be 235 kDa. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single polypeptide was found with a molecular mass of 127 kDa. From the pyridine hemochrome spectrum 1 protoheme IX/polypeptide was calculated. The light absorbance bands of the dithionite-reduced cytochrome were found to be at 558.5 (alpha), 529 (beta), and 426 nm (Soret), and that of the oxidized cytochrome at 413.5 nm. The difference absorbance coefficients are delta epsilon (426.5 - 440 nm) = 160.6 +/- 11 mM-1 cm-1 and delta epsilon (558.5 - 542 nm) = 29.3 +/- 2 mM-1 cm-1 (mean +/- S.D.; n = 5). Carbon monoxide binds to the cytochrome in a time-dependent fashion (maximum binding after 50-60 min). The midpoint potential of the solubilized nonpurified cytochrome is identical to the cytochrome in situ (Em7.0 = -218 +/- 7 mV (mean +/- S.D.; n = 5)). However, purified cytochrome b shows a significantly decreased midpoint potential, estimated at -407 +/- 18 mV (n = 4). The protein does not contain noncovalently bound FAD or FMN, and no spectral evidence was obtained for the presence of covalently bound flavin. Preliminary amino acid analysis of the cytochrome shows a high content of hydrophilic residues.     

The acceptor quinone complex of Rhodopseudomonas viridis reaction centers

The acceptor complex of isolated reaction centers from Rhodopseudomonas viridis contains both menaquinone and ubiquinone. In a series of flashes the ubiquinone was observed to undergo binary oscillations in the formation and disappearance of a semiquinone, indicative of secondary acceptor (QB) activity. The oscillating signal, Q-B, was typical of a ubisemiquinone anion with a peak at 450 nm (delta epsilon = 6 mM-1 X cm-1) and a shoulder at 430 nm. Weak electrochromic bandshifts in the infrared were also evident. The spectrum of the reduced primary acceptor (Q-A) exhibited a major peak at 412 nm (delta epsilon = 10 mM-1 X cm-1) consistent with the assignment of menaquinone as QA. The Q-A spectrum also had minor peaks at 385 and 455 nm in the blue region. The same spectrum was recorded after quantitative removal of the secondary acceptor, when only menaquinone was present in the reaction centers. Spectral features in the near-infrared due to Q-A were attributed to electrochromic effects on bacteriochlorophyll (BChl) b and bacteriopheophytin (BPh) b pigments resulting in a distinctive split peak at 810 and 830 nm (delta epsilon = 8 mM-1 X cm-1). The menaquinone was identified as 2-methyl-3-nonylisoprenyl-1,4-naphthoquinone (menaquinone-9). The native QA activity was uniquely provided by this menaquinone and ubiquinone was not involved. QB activity, on the other hand, displayed at least a 40-fold preference for ubiquinone (Q-10) as compared to menaquinone. Thus, both quinone-binding sites display remarkable specificity for their respective quinones. In the absence of donors to P+, charge recombination of the P+Q-A and P+Q-B pairs had half-times of 1.1 +/- 0.2 and 110 +/- 20 ms, respectively, at pH 9.0, indicating an electron-transfer equilibrium constant (Kapp2) of at least 100 for Q-AQB in equilibrium QAQ-B. Also observed was a slow recombination of the cytochrome c-558+ Q-A pair, with t 1/2 = 2 +/- 0.5 s at pH 6.     

A near-infrared investigation of cytochrome c oxidase in higher plant mitochondria

Optical features of cytochrome c oxidase in potato mitochondria have been characterized in the near-ir region. In order to discriminate the respective properties of the various redox centers, the redox state was monitored from free and inhibited, bound species. Appropriate comparisons singled out difference spectra which can be attributed specifically to CuA and CuB. The CuA difference spectrum (red-ox) exhibits a negative band centered at 812 nm and, analogous to its mammalian counterpart, the so-called 830-nm band (delta epsilon red/ox = -2.0 mM-1 cm-1). The unusual difference spectrum (red-ox) assigned to CuB is characterized by a broad positive band also centered at 812 nm with an extinction coefficient of delta epsilon red/ox = 4.3 mM-1 cm-1.     

Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens

Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 +/- 3 kDa). Its pI was 6.05. The EPR spectrum showed an axial signal g at 2.21(8) and g at 2.04(5). The magnitude of the hyperfine splitting (A parallel = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (epsilon = 7.0 mM-1 cm-1), and a broad shoulder around 780 nm. A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (epsilon = 2.5 mM-1 cm-1) and pronounced structures in the ultraviolet region. The EPR parameters were g parallel = 2.26(6) and g perpendicular = 2.05(6), with A parallel = 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide. The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 mumol substrate min-1 mg protein-1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite. No 'oxidase' activity could be demonstrated for the enzyme. Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomonas.     

Purification and biochemical characterization of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough).

A novel iron-sulfur protein has been isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. Analysis of the metal and acid-labile sulfur content of the protein revealed the presence of 6.3 +/- 0.4 Fe/polypeptide and 6.2 +/- 0.7 S2-/polypeptide. Non-iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S = 1/2 [6Fe-6S]3+ prismane-like EPR signal in the dithionite-reduced protein, we believe that we have encountered the first example of a prismane-cluster-containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI = 4.9). The ultraviolet/visible spectrum is relatively featureless (epsilon 280 = 81 mM-1.cm-1, epsilon 400 = 25 mM-1.cm-1, epsilon 400,red = 14 mM-1.cm-1). The shape of the protein is approximately globular (S20.w = 4.18 S). The N-terminal amino acid sequence is MFS/CFQS/C QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross-reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.     

Characterization of indo-1 and quin-2 as spectroscopic probes for Zn2(+)-protein interactions

1-[2-Amino-5-(6-carboxyindol-2-yl)phenoxyl]-2-(2'- amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (indo-1) and 2-[2-(bis(carboxymethyl)amino-5-methylphenoxy) methyl]-6- methyl-8-[bis-(carboxymethyl)amino]quinoline (quin-2) are sensitive, spectral indicators for Zn2+. Additions of subsaturating Zn2+ to 10-80 microM indo-1 or quin-2 at pH 7.0 produce uv difference spectra with isosbestic wavelengths at 342 and 282 nm or at 342, 317, and 252 nm, respectively. Formation of 1:1 Zn2+:indicator complexes at pH 7.0 and 20 degrees C in the absence (presence) of 100 mM KCl gives delta epsilon max = -2.4 +/- 0.2 X 10(4) M-1 cm-1 at 367 nm (-2.1 +/- 0.2 X 10(4) M-1 cm-1 at 365 nm) for indo-1 and delta epsilon max = -2.7 +/- 0.1 X 10(4) M-1 cm-1 at 266 nm (-2.6 +/- 0.1 X 10(4) M-1 cm-1 at 265 nm) for quin-2. Competition experiments at pH 7.0 and 20 degrees C with indo-1 and quin-2 and also 4-(2-pyridylazo)resorcinol (PAR) as the second chelator in the absence (presence) of 100 mM KCl yield apparent affinity constants: K'A = 2.5 +/- 1.0 X 10(10) M-1 (6.2 +/- 0.5 X 10(9) M-1) for indo-1 binding Zn2+ and K'A = 9.4 +/- 3.3 X 10(11) M-1 (2.7 +/- 0.1 X 10(11) M-1) for quin-2 binding Zn2+. The above constants provide the basis for rapid steady-state spectrophotometric determinations of the affinity of a protein for Zn2+ with K'A approximately 10(10) - 10(13) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic and structural characterization of cis-trans isomerization of 12-(S)-hydroxy-(5Z, 8E, 10E)-heptadecatrienoic acid by high-performance liquid chromatography and gaschromatography-mass spectrometry.

It is shown that 12-(S)-hydroxy-(5Z, 8E, 10E)-heptadecatrienoic acid (5-cis-HHT)--a physiological metabolite of arachidonic acid--is acid-catalyzed converted into a less polar substance with its maximum UV-absorption at (1)max=232 nm and a molar absorptivity of about epsilon=26600 +/- 200 M(-1)cm(-1). Using a reversed-phase high-performance liquid chromatographic (HPLC) method this equilibrium reaction (K(c) = 1.78 +/- 0.05 at pH 1.10 and 298 K) could be thermodynamicly characterized as a pH dependent, exergonic and exothermic reaction according to kinetics of a first order reaction (at pH 1.10 and 298 K: delta(R)G(o) = -1.42 +/- 0.07 kJ mol(-1), delta(R)H(o) = -3.50 +/- 0.9 kJ mol(-1), delta(R)S(o) = 7.0 +/- 3.0 J mol(-1)*K, delta(R)H*f = 100.0 +/- 4.0 kJ mol(-1)). Kinetic data for several pH-values and temperatures are presented. These data and structural characterization by gaschromatography-mass spectrometry (GC/MS) lead to the conclusion that 5-cis-HHT is isomerized to 12-(S)-hydroxy-(5E, 8E, 10E)-heptadecatrienoic acid (5-trans-HHT).     

Identity of the subunits and the stoicheiometry of prosthetic groups in trimethylamine dehydrogenase and dimethylamine dehydrogenase.

Trimethylamine dehydrogenases from bacterium W3A1 and Hyphomicrobium X and the dimethylamine dehydrogenase from Hyphomicrobium X were found to contain only one kind of subunit. The millimolar absorption coefficient of a single [4Fe-4S] cluster in trimethylamine dehydrogenase from bacterium W3A1 was estimated to be 14.8 mM-1 . cm-1 at 443 nm. From this value a 1:1 stoicheiometry of the prosthetic groups, 6-S-cysteinyl-FMN and the [4Fe-4S] cluster, was established. Millimolar absorption coefficients of the three enzymes were in the range 49.4-58.7 mM-1 . cm-1 at approx. 440 nm. This range of values is consistent with the presence of two [4Fe-4S] clusters and two flavin residues, for which the millimolar absorption coefficient had earlier been found to be 12.3 mM-1 . cm-1 at 437 nm. The N-terminal amino acid was alanine in each of the three enzymes. Sequence analysis of the first 15 residues from the N-terminus of dimethylamine dehydrogenase indicated a single unique sequence. Two identical subunits, each containing covalently bound 6-S-cysteinyl-FMN and a [4Fe-4S] cluster, in each of the enzymes are therefore indicated.     

Azide-binding studies reveal type 3 copper heterogeneity in ascorbate oxidase from the green zucchini squash (Cucurbita pepo).

Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with 'high affinity' (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with 'low affinity' (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.     

Coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Protection by the substrate from inactivation by light

Partially purified 2-methyleneglutarate mutase from Clostridium barkeri was separated from 3-methylitaconate delta-isomerase by treatment with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) followed by FPLC on the anion exchange column Mono Q in the presence of the detergent. When purified in the dark, the active mutase contained a corrinoid, most probably coenzyme B12. The enzyme was inactivated by light at the same wavelength (lambda less than 620 nm) and rate as free coenzyme B12. The rate was not influenced by oxygen or by temperature (0-37 degrees C). Reactivation of up to 50% of the original activity was achieved by incubation with coenzyme B12 and dithiothreitol. The substrates 2-methyleneglutarate (up to 40 mM) or (R)-3-methylitaconate specifically protected the enzyme from inactivation by visible light. This effect was enhanced 3-fold by raising the temperature from 0 degrees C to 37 degrees C. The data indicate that during catalysis, the Co-C bond of the coenzyme is cleaved and cannot be affected any more by light.     

6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin

The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C. While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1). Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C. The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C. In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin. The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm. Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.     

An investigation of the equilibria between aqueous ribose, ribulose, and arabinose

The thermodynamics of the equilibria between aqueous ribose, ribulose, and arabinose were investigated using high-pressure liquid chromatography and microcalorimetry. The reactions were carried out in aqueous phosphate buffer over the pH range 6.8-7.4 and over the temperature range 313.15-343.75 K using solubilized glucose isomerase with either Mg(NO3)2 or MgSO4 as cofactors. The equilibrium constants (K) and the standard state Gibbs energy (delta G degrees) and enthalpy (delta H degrees) changes at 298.15 K for the three equilibria investigated were found to be: ribose(aq) = ribulose(aq) K = 0.317, delta G degrees = 2.85 +/- 0.14 kJ mol-1, delta H degrees = 11.0 +/- 1.5 kJ mol-1; ribose(aq) = arabinose(aq) K = 4.00, delta G degrees = -3.44 +/- 0.30 kJ mol-1, delta H degrees = -9.8 +/- 3.0 kJ mol-1; ribulose(aq) = arabinose(aq) K = 12.6, delta G degrees = -6.29 +/- 0.34 kJ mol-1, delta H degrees = -20.75 +/- 3.4 kJ mol-1. Information on rates of the above reactions was also obtained. The temperature dependencies of the equilibrium constants are conveniently expressed as R in K = -delta G degrees 298.15/298.15 + delta H degrees 298.15[(1/298.15)-(1/T)] where R is the gas constant (8.31441 J mol-1 K-1) and T the thermodynamic temperature.     

M?ssbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas. A ferrous heme with an unusually large quadrupole splitting

Hydroxylamine oxidoreductase from Nitrosomonas europeae catalyzes the oxidative conversion of NH2OH to NO-2. The enzyme, Mr = 220,000, has an (alpha beta)3 subunit structure with each alpha beta subunit containing 7-8 c-type hemes and one unusual prosthetic group, termed P-460. The P-460 is also found in a Mr approximately equal to 17,000 protein (P-460 fragment). M?ssbauer spectra of the reduced P-460 groups, in hydroxylamine oxidoreductase and the fragment, exhibit nearly identical quadrupole doublets with an unusually large splitting, delta EQ = 4.21 mm/s (no ferrous heme protein is known with delta EQ greater than 2.75 mm/s). The observed isomer shift, delta = 0.96 mm/s at 4.2 K, shows that the P-460 iron is high spin ferrous. Treatment of oxidized hydroxylamine oxidoreductase with H2O2 followed by reduction or exposure of the native sample to CO led to the disappearance of both the characteristic 460 nm absorption band (epsilon = 89 mM-1 cm-1) and the delta EQ = 4.21 mm/s doublet. The iron of the oxidized P-460 fragment is high spin ferric, with M?ssbauer and EPR parameters very similar to those of metmyoglobin. Optical spectra of the reduced P-460 fragment show long wavelength bands at 650 and 688 nm which are sensitive to treatment of the fragment with reagents which react with P-460. These bands were, however, not detected in hydroxylamine oxidoreductase. The spectroscopic and chemical evidence obtained to date suggests strongly that the P-460 iron resides in a heme-like macrocycle although the presumed porphyrin must have some unusual features.     

The Type 3 copper site is intact but labile in Type 2-depleted laccase

We report results of experiments designed to characterize the Type 1 and Type 3 copper sites in Rhus laccase depleted of Type 2 copper (T2D). Use of the Lowry method for determining protein concentration yielded the value 5620 +/- 570 M-1 cm-1 for the extinction of the 615-nm absorption band of this protein. Anaerobic reductive titrations with Ru(NH)3)6(2)+ and Cr(II)aq ions established the presence of three electron-accepting centers, which are reduced in a complex manner. Treatment of T2D laccase with a 70-fold excess of H2O2 induced a new shoulder at 330 nm (delta epsilon = 660 M-1 cm-1), as well as intensity perturbations at 280 and 615 nm. Comparison of difference spectra show that this 330-nm band derives from a Type 3 copper-bound peroxide and not from a reoxidized Type 3 site. Dioxygen reoxidation of ascorbate-reduced T2D laccase produced new difference bands at 330 nm (delta epsilon = 770 M-1 cm-1) and 270 nm (delta epsilon = 13,000 M-1 cm-1), the former assigned to a bound peroxide which is a dioxygen reduction intermediate. In the corresponding epr spectrum of this material new Cu(II) g parallel features (A parallel approximately 130 G) indicative of an isolated copper ion and a triplet signal near 3,400 G were observed, originating from the Type 3 sites of separate T2D laccase molecules. Reoxidation by ferricyanide or by dioxygen as mediated by iron hexacyanide did not produce these changes. Thus the magnetism of the reoxidized Type 3 site in T2D laccase can be perturbed as a consequence of aerobic turnover. The suggestion is advanced that there are presently three forms of T2D laccase, possibly metastable conformational isotypes, accounting for the apparently contradictory reports on the properties of this protein.     

Isolation and characterization of an Fe,-S8 ferredoxin (ferredoxin II) from Clostridium thermoaceticum.

A second ferredoxin protein was isolated from the thermophilic anaerobic bacterium Clostridium thermoaceticum and termed ferredoxin II. This ferredoxin was found to contain 7.9 +/- 0.3 iron atoms and 7.4 +/- 0.4 acid-labile sulfur atoms per mol of protein. Extrusion studies of the iron-sulfur centers showed the presence of two [Fe4-S4] centers per mol of protein and accounted for all of the iron present. The absorption spectrum was characterized by maxima at 390 nm (epsilon 390 = 30,400 M-1cm-1) and 280 nm (epsilon 280 = 41.400 M-1 cm-1) and by a shoulder at 300 nm. The ration of the absorbance of the pure protein at 390 nm to the absorbance at 280 nm was 0.74. Electron paramagnetic resonance data showed a weak signal in the oxidized state, and the reduced ferredoxin exhibited a spectrum typical of [Fe4-S4] clusters. Double integration of the reduced spectra showed that two electrons were necessary for the complete reduction of ferredoxin II. Amino histidine, and 1 arginine, and a molecular weight of 6,748 for the native protein. The ferredoxin is stable under anaerobic conditions for 60 min at 70 degrees C. The average oxidation-reduction potential for the two [Fe4-S4] centers was measured as -365 mV.     

Purification and Characterization of Maleate Hydratase from Pseudomonas pseudoalcaligenes

Maleate hydratase (malease) from Pseudomonas pseudoalcaligenes has been purified. The purified enzyme (98% pure) catalyzes the stereospecific addition of water to maleate and citraconate (2-methylmaleate), forming d-(+)-malate and d-(+)-citramalate, respectively. 2,3-Dimethylmaleate was also a substrate for malease. The stability of the enzyme was dependent on the protein concentration and the addition of dicarboxylic acids. The purified enzyme (89 kDa) consisted of two subunits (57 and 24 kDa). No cofactor was required for full activity of this colorless enzyme. Maximum enzyme activity was measured at pH 8 and 45 degrees C. The K(m) for maleate was 0.35 mM, and that for citraconate was 0.20 mM. Thiol reagents, such as p-chloromercuribenzoate and iodoacetamide, and sodium dodecyl sulfate completely inhibited malease activity. Malease activity was competitively inhibited by d-malate (K(i) = 0.63 mM) and d-citramalate (K(i) = 0.083 mM) and by the substrate analog 2,2-dimethylsuccinate (K(i) = 0.025 mM). The apparent equilibrium constants for the maleate, citraconate, and 2,3-dimethylmaleate hydration reactions were 2,050, 104, and 11.2, respectively.     

Characterisation and quantitation of a selenol intermediate in the reaction of ebselen with thiols.

The reaction of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) with thiols was investigated with particular attention to the formation of an ebselen selenol intermediate. The selenol intermediate could be trapped in a mixture of ebselen and thiols with 1-chloro-2,4-dinitrobenzene and the resulting product displayed unique spectral characteristics. The reaction of authentic, synthesised ebselen selenol with 1-chloro-2,4-dinitrobenzene (CDNB) was shown to give rise to the same compound (2,4-dinitrophenyl (N-phenyl-2-carboxamido phenyl) selenide as characterized by light spectroscopy, NMR, IR and elemental analysis. The determination of the absorbtion coefficient at 400 nm (E = 7.5 mM-1 cm-1) and the initial rate constant of the reaction (1.4 +/- 0.3 mM-1 min-1) allows for the convenient quantification of ebselen selenol concentrations by initial rate measurements after addition of CDNB. The choice of 400 nm to monitor the reaction excludes the interference of other intermediates in the reaction of ebselen with thiols as well as the reaction of the thiols with CDNB. When the assay is applied to typical incubation conditions used for investigating the glutathione peroxidase-like activity of ebselen it was shown that as much as 10-20% of ebselen is in the selenol form. If a stronger reductant (dithiothreitol) is used 60% is in the selenol form. These data could also be confirmed by the direct determination of ebselen selenol by UV spectroscopy, due to its peak absorption at 370 nm (E = 2 mM-1 cm-1). In conclusion, this investigation demonstrates, for the first time, the identity and quantity of ebselen selenol in the reaction of ebselen with thiols and also describes a convenient assay for its quantification. These observations allow further possibilities for investigation of the molecular species responsible for the antioxidant and peroxidase activities of ebselen.     

A study of vibrational relaxation of B-state carbon monoxide in the heme pocket of photolyzed carboxymyoglobin.

The vibrational energy relaxation of dissociated carbon monoxide in the heme pocket of sperm whale myoglobin has been studied using equilibrium molecular dynamics simulation and normal mode analysis methods. Molecular dynamics trajectories of solvated myoglobin were run at 300 K for both the delta- and epsilon-tautomers of the distal histidine, His64. Vibrational population relaxation times were estimated using the Landau-Teller model. For carbon monoxide (CO) in the myoglobin epsilon-tautomer, for a frequency of omega0 = 2131 cm-1 corresponding to the B1 state, T1epsilon(B1) = 640 +/- 185 ps, and for a frequency of omega0 = 2119 cm-1 corresponding to the B2 state, T1epsilon(B2) = 590 +/- 175 ps. Although the CO relaxation rates in both the epsilon- and delta-tautomers are similar in magnitude, the simulations predict that the vibrational relaxation of the CO is faster in the delta-tautomer. For CO in the myoglobin delta-tautomer, it was found that the relaxation times were identical within error for the two CO substate frequencies, T1delta(B1) = 335 +/- 115 ps and T1delta(B2) = 330 +/- 145 ps. These simulation results are in reasonable agreement with experimental results of Anfinrud and coworkers (unpublished results). Normal mode calculations were used to identify the dominant coupling between the protein and CO molecules. The calculations suggest that the residues of the myoglobin pocket, acting as a first solvation shell to the CO molecule, contribute the primary "doorway" modes in the vibrational relaxation of the oscillator.     

p10 single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39

The RNA binding protein of 56 residues encoded by the extreme 3' region of the gag gene of Rauscher murine leukemia virus (MuLV) has been chemically synthesized by a solid-phase synthesis approach. Since the peptide contains a Cys26-X2-Cys29-X4-His34-X2-Cys39 sequence that is shared by all retroviral gag polyproteins which has been proposed to be a metal binding region, it was of considerable interest to examine the metal binding properties of the complete p10 protein. As postulated, p10 binds the metal ions Cd(II), Co(II), and Zn(II). The Co(II) protein shows a set of d-d absorption bands typical of a tetrahedral Co(II) complex at 695 (epsilon = 565 M-1 cm-1), 642 (epsilon = 655 M-1 cm-1), and 615 nm (epsilon = 510 M-1 cm-1) and two intense bands at 349 (epsilon = 2460 M-1 cm-1) and 314 nm (epsilon = 4240 M-1 cm-1) typical of Co(II)----(-)S- charge transfer. The ultraviolet absorption spectrum also indicates Cd(II) binding by the appearance of a Cd(II)----(-)S- charge-transfer band at 255 nm. The 113Cd NMR spectrum of 113Cd(II)-p10 reveals one signal at delta = 648 ppm. This chemical shift correlates well with that predicted for ligation of 113Cd(II) to three -S- from the three Cys residues of p10. The chemical shift of 113Cd(II)-p10 changes by only 4 ppm upon binding of d(pA)6, indicating that the chelate complex is little changed by oligonucleotide binding.(ABSTRACT TRUNCATED AT 250 WORDS)