The effect of substrate, dihydrobiopterin, and dopamine on the EPR spectroscopic properties and the midpoint potential of the catalytic iron in recombinant human phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH(4)) and non-heme iron-dependent enzyme that hydroxylates L-Phe to L-Tyr. The paramagnetic ferric iron at the active site of recombinant human PAH (hPAH) and its midpoint potential at pH 7.25 (E(m)(Fe(III)/Fe(II))) were studied by EPR spectroscopy. Similar EPR spectra were obtained for the tetrameric wild-type (wt-hPAH) and the dimeric truncated hPAH(Gly(103)-Gln(428)) corresponding to the "catalytic domain." A rhombic high spin Fe(III) signal with a g value of 4.3 dominates the EPR spectra at 3.6 K of both enzyme forms. An E(m) = +207 +/- 10 mV was measured for the iron in wt-hPAH, which seems to be adequate for a thermodynamically feasible electron transfer from BH(4) (E(m) (quinonoid-BH(2)/BH(4)) = +174 mV). The broad EPR features from g = 9.7-4.3 in the spectra of the ligand-free enzyme decreased in intensity upon the addition of L-Phe, whereas more axial type signals were observed upon binding of 7,8-dihydrobiopterin (BH(2)), the stable oxidized form of BH(4), and of dopamine. All three ligands induced a decrease in the E(m) value of the iron to +123 +/- 4 mV (L-Phe), +110 +/- 20 mV (BH(2)), and -8 +/- 9 mV (dopamine). On the basis of these data we have calculated that the binding affinities of L-Phe, BH(2), and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the enzyme, with respect to the ferric form. Interestingly, an E(m) value comparable with that of the ligand-free, resting form of wt-hPAH, i.e. +191 +/- 11 mV, was measured upon the simultaneous binding of both L-Phe and BH(2), representing an inactive model for the iron environment under turnover conditions. Our findings provide new information on the redox properties of the active site iron relevant for the understanding of the reductive activation of the enzyme and the catalytic mechanism.     

Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli

The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.     

Human glutathione transferase A1-1 demonstrates both half-of-the-sites and all-of-the-sites reactivity

A study of the kinetics of a heterodimeric variant of glutathione transferase (GST) A1-1 has led to the conclusion that, although the wild-type enzyme displays all-of-the-sites reactivity in nucleophilic aromatic substitution reactions, it demonstrates half-of-the-sites reactivity in addition reactions. The heterodimer, designed to be essentially catalytically inactive in one subunit due to a single point mutation (D101K), and the two parental homodimers were analyzed with seven different substrates, exemplifying three types of reactions catalyzed by glutathione transferases (nucleophilic aromatic substitution, addition, and double-bond isomerization reactions). Stopped-flow kinetic results suggested that the wild-type GST A1-1 behaved with half-of-the-sites reactivity in a nucleophilic aromatic substitution reaction, but steady-state kinetic analyses of the GST A1-D101K heterodimer revealed that this was presumably due to changes to the extinction coefficient of the enzyme-bound product. In contrast, steady-state kinetic analysis of the heterodimer with three different substrates of addition reactions provided evidence that the wild-type enzyme displayed half-of-the-sites reactivity in association with these reactions. The half-of-the-sites reactivity was shown not to be dependent on substrate size, the level of saturation of the enzyme with glutathione, or relative catalytic rate.     

High-performance liquid chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide.

The functional importance of glycolipids has emphasized the need for more sensitive methods of detection, characterization, and quantification than has often been possible using traditional thin-layer chromatographic techniques. We describe the use of ceramide glycanase and HPLC to identify and quantify gangliosides in which the carbohydrate is in Glcbeta1--> linkage with ceramide. Detection of released carbohydrate was by fluorescent labeling with 2-aminobenzamide at the reducing terminal prior to HPLC analysis. Under the conditions described, ceramide glycanase hydrolyzed all of the common gangliosides studied, offering a broad spectrum of specificity. Release and detection of carbohydrate were linear over a wide range (over two orders of magnitude) of micromolar glycolipid substrate concentrations. Use of an N-linked glycan as an internal standard allowed accurate quantification and a recovery of 93% was achieved. The method additionally maintained the sensitivity (chromatographic peaks containing 1 pmol were readily detected from tissue samples) and comparable resolution to related assays. This was shown by the separation, not only of isomeric carbohydrates from the "a" and "b" series, but also of ganglioside carbohydrate differing only by the presence of either N-acetyl- or N-glycolylneuraminic acid. Application of the method to neutral glycosphingolipids and to tissue samples, including 10-microl quantities of plasma, is illustrated. Glycan structures were confirmed by exoglycosidase digestion and/or matrix-assisted laser desorption/ionization mass spectrometry.     

Inhibition of activated/memory (CD45RO(+)) T cells by oxidative stress associated with block of NF-kappaB activation

Impaired immune responses in cancer patients have been associated with oxidative stress. Increased levels of reactive oxygen species released from activated, tumor-infiltrating macrophages or granulocytes may therefore constitute a hurdle for effective immunotherapy against cancer. In this study, we investigated functional consequences and molecular events in T cells exposed to low levels of oxidative stress. We observed that cytokine production of human PBMC, upon stimulation with an HLA-A*0201-restricted influenza peptide and nonspecific receptor cross-linking, was reduced after exposure to micromolar levels of H2O2. Functional impairment as measured by IFN-gamma release occurred earlier and at lower doses of exogenously added H2O2 than required to induce apoptosis. This suggests that there is a dose window of oxidative stress leading to T cell unresponsiveness in the absence of apoptosis. The reduction of Th1 cytokines, induced by H2O2, was predominantly observed in memory/effector (CD45RO(+)) T cells and correlated with a block in NF-kappaB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-gamma, TNF-alpha, and IL-2. The influence of H2O2 on production of IL-10 was not significantly different between memory/activated and naive T cells. These observations suggest that Th1 and Th2 cytokines are differently regulated under conditions of oxidative stress. Taken together, these findings may explain why Ag-experienced, CD45RO(+), T cells found in the tumor milieu are functionally suppressed.     

Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol

Infants with the cholesterol synthesis defect Smith- Lemli-Opitz syndrome (SLO) have reduced activity of the enzyme 7-dehydrocholesterol-7-reductase and accumulate 7-dehydrocholesterol, with the highest concentration in the brain. As a result of the generally reduced content of cholesterol, plasma levels of oxysterols would be expected to be reduced. 24S-hydroxycholesterol is almost exclusively formed in the brain, whereas 27-hydroxycholesterol is mainly formed from extrahepatic and extracerebral cholesterol. In accordance with the expectations, sterol-correlated plasma levels of 24S-hydroxycholesterol were reduced in infants with SLO (by about 50%). In contrast, the sterol-correlated levels of 27-hydroxycholesterol in the circulation were markedly increased. No side-chain oxidized metabolites of 7-dehydrocholesterol were detected in the circulation. Recombinant human CYP27 had markedly lower 27-hydroxylase activity toward 7-dehydrocholesterol than towards cholesterol. HEK293 cells expressing 24S-hydroxylase active toward cholesterol had no significant activity towards 7-dehydrocholesterol. The plasma levels of 3 beta,7 alpha-dihydroxy-5-cholestenoic in the patients acid were reduced, suggesting a generally reduced metabolism of 27-oxygenated steroids. It is concluded that the accumulation of 7-dehydrocholesterol in the brains of patients with SLO reduces formation of 24S-hydroxycholesterol. The condition is associated with markedly increased circulating levels of 27-hydroxycholesterol, most probably due to reduced metabolism of this oxysterol. We discuss the possibility that the circulating levels of 24S-hydroxycholesterol may be used as a marker for the severity of the disease.--Bj?rkhem, I., L. Starck, U. Andersson, D. Lütjohann, S. von Bahr, I. Pikuleva, A. Babiker, and U. Diczfaulsy. Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol. J. Lipid Res. 2001. 42: 366--371.     

Chloroplast biogenesis. Regulation of lipid transport to the thylakoid in chloroplasts isolated from expanding and fully expanded leaves of pea

To study the regulation of lipid transport from the chloroplast envelope to the thylakoid, intact chloroplasts, isolated from fully expanded or still-expanding pea (Pisum sativum) leaves, were incubated with radiolabeled lipid precursors and thylakoid membranes subsequently were isolated. Incubation with UDP[(3)H]Gal labeled monogalactosyldiacylglycerol in both envelope membranes and digalactosyldiacylglycerol in the outer chloroplast envelope. Galactolipid synthesis increased with incubation temperature. Transport to the thylakoid was slow below 12 degrees C, and exhibited a temperature dependency closely resembling that for the previously reported appearance and disappearance of vesicles in the stroma (D.J. Morré, G. Selldén, C. Sundqvist, A.S. Sandelius [1991] Plant Physiol 97: 1558-1564). In mature chloroplasts, monogalactosyldiacylglycerol transport to the thylakoid was up to three times higher than digalactosyldiacylglycerol transport, whereas the difference was markedly lower in developing chloroplasts. Incubation of chloroplasts with [(14)C]acyl-coenzyme A labeled phosphatidylcholine (PC) and free fatty acids in the inner envelope membrane and phosphatidylglycerol at the chloroplast surface. PC and phosphatidylglycerol were preferentially transported to the thylakoid. Analysis of lipid composition revealed that the thylakoid contained approximately 20% of the chloroplast PC. Our results demonstrate that lipids synthesized at the chloroplast surface as well as in the inner envelope membrane are transported to the thylakoid and that lipid sorting is involved in the process. Furthermore, the results also indicate that more than one pathway exists for galactolipid transfer from the chloroplast envelope to the thylakoid.     

Active oxygen produced during selective excitation of photosystem I is damaging not only to photosystem I, but also to photosystem II

With the aim to specifically study the molecular mechanisms behind photoinhibition of photosystem I, stacked spinach (Spinacia oleracea) thylakoids were irradiated at 4 degrees C with far-red light (>715 nm) exciting photosystem I, but not photosystem II. Selective excitation of photosystem I by far-red light for 130 min resulted in a 40% inactivation of photosystem I. It is surprising that this treatment also caused up to 90% damage to photosystem II. This suggests that active oxygen produced at the reducing side of photosystem I is highly damaging to photosystem II. Only a small pool of the D1-protein was degraded. However, most of the D1-protein was modified to a slightly higher molecular mass, indicative of a damage-induced conformational change. The far-red illumination was also performed using destacked and randomized thylakoids in which the distance between the photosystems is shorter. Upon 130 min of illumination, photosystem I showed an approximate 40% inactivation as in stacked thylakoids. In contrast, photosystem II only showed 40% inactivation in destacked and randomized thylakoids, less than one-half of the inactivation observed using stacked thylakoids. In accordance with this, photosystem II, but not photosystem I is more protected from photoinhibition in destacked thylakoids. Addition of active oxygen scavengers during the far-red photosystem I illumination demonstrated superoxide to be a major cause of damage to photosystem I, whereas photosystem II was damaged mainly by superoxide and hydrogen peroxide.     

Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum

Bovine muscle carbonic anhydrase (isoenzyme III; BCAIII) exhibited a three-state unfolding process at equilibrium upon denaturation in guanidine hydrochloride (GuHCl). The stable folding intermediate appeared to be of molten globule type. The stability towards GuHCl in terms of mid-point concentrations of denaturation were very similar for BCAIII and human CAII (HCAII). It was further demonstrated that the aromatic amino acid residues contributed significantly to the circular dichroism (CD) spectrum in the far-UV wavelength region during the native-->molten globule state transition. Thus, the ellipiticity change at 218 nm was shown to monitor the loss of tertiary interactions of aromatic side chains at the first unfolding transition as well as the rupture of secondary structure at the second unfolding transition. Similar aromatic contributions to the far-UV CD spectrum, but with varying magnitudes, were also noted for BCAII and HCAII, further emphasizing that interference of aromatic residues should not be neglected at wavelengths that normally are assigned to secondary structural changes.     

A revised model of the active site of alternative oxidase

The plant mitochondrial protein alternative oxidase catalyses dioxygen dependent ubiquinol oxidation to yield ubiquinone and water. A structure of this protein has previously been proposed based on an assumed structural homology to the di-iron carboxylate family of proteins. However, these authors suggested the protein has a very different topology than the known structures of di-iron carboxylate proteins. We have re-examined this model and based on comparison of recent sequences and structural data on di-iron carboxylate proteins we present a new model of the alternative oxidase which allows prediction of active site residues and a possible membrane binding motif.