Proton nuclear magnetic resonance studies of compounds I and II of horseradish peroxidase.

Ter-butyl hydroperoxide (TBH) induced microsomal lipid peroxidation has been measured by oxygen consumption and malonaldehyde (MDA) formation. It has been found that the singlet oxygen (¹O₂) trap 2,5 diphenylfuran depressed both oxygen consumption and MDA formation. In contrast, histidine, another ¹O₂ trap does not effect neither oxygen consumption, nor MDA production. On the other hand, β-carotene, a ¹O₂ quencher strongly depresses oxygen consumption but slightly affects MDA production. Such results are consistent with the generation of ¹O₂ as transient by product of peroxidative microsomal lipid decomposition.     

Proton nuclear magnetic resonance studies on the iodide binding by horseradish peroxidase

Binding of an iodide ion to horseradish peroxidase was studied by following the hyperfine-shifted proton nuclear magnetic resonance signals of the enzyme. For the enzyme in an iodide-free solution, the spectra of hyperfine-shifted methyl region were only slightly affected by varying pH. In the presence of iodide (200 mM), however, both chemical shifts and line widths of the heme peripheral 1- and 8-methyl proton signals were markedly affected by the pH change from 7 to 4 and broadened at pH 4. From the change in peak heights of these signals at various concentrations of iodide, the dissociation constant of the iodide to the enzyme was calculated to be about 100 mM at pH 4.0. The peak derived from the proximal histidyl imidazole N epsilon-H proton was not perturbed by the addition of 200 mM iodide at pH 4.0 and 7.1. The rate of oxidation of iodide with hydrogen peroxide catalyzed by the enzyme was increased with decreasing pH, indicating the participation of an ionizable group with the pKa value of 4.0. Optical difference spectrum studies showed that iodide exerts no effect both at pH 4.0 and 7.4 on the binding affinity of resorcinol which is associated with the enzyme in the vicinity of the heme peripheral 8-CH3 group. These results suggest that an iodide ion binds to the enzyme at almost equal distance from the heme peripheral 1- and 8-methyl groups at the distal side of the heme and that the interaction becomes stronger in acidic medium with protonation of the ionizable group with the pKa value of 4.0.     

Proton nuclear magnetic resonance spectroscopy of horseradish peroxidase isoenzymes: correlation of distinctive spectra with isoenzyme specific activities

High-resolution proton NMR spectra are reported for the paramagnetic ferric native and cyano complexes of the five major horseradish root peroxidase (HRP) isoenzymes (A1, A2, A3, B, and C). Axial imidazole resonances are observed in the native and cyano-complex spectra of all the isoenzymes, thus indicating the presence of a common axial histidine ligand. Proton NMR spectra outside the usual diamagnetic region are identical for sets of A1 and A2 isoenzymes and for the B and C isoenzyme set. Variation in heme residue chemical shift positions may be controlled in part by porphyrin vinyl side chain-protein interactions. Diverse upfield spectra among the isoenzymes reflect amino acid substitutions and/or conformational differences near the prosthetic group, as signals in this region must result from amino acid residues in proximity to the heme center. Acid-base dependence studies reveal an "alkaline" transition that converts the native high-spin iron (III) porphyrin to the low-spin state. The transition occurs at pH 9.3, 9.4, 9.8, and 10.9 for respective HRP A1, A2, A3, and C isoenzymes, respectively. Significantly, this ordering also reflects specific activities for the isoenzymes in the order A1 = A2 greater than A3 greater than B = C. Identical proton NMR spectra for A1/A2 and B/C isoenzyme sets parallel equivalent specific activities for members of a particular set. Proton NMR spectra thus appear to be highly sensitive to protein modifications that affect catalytic activity.     

Proton nuclear magnetic resonance spectra of Pseudomonas aeruginosa ferricytochrome cd1

Proton nuclear magnetic resonance spectra of ferricytochrome cd1 from the denitrifying bacterium Pseudomonas aeruginosa have been obtained. The normal 0-10-ppm chemical shift range shows many overlapping and nonresolvable peaks, as would be expected for a dimeric protein of molecular weight approximately 120,000. In the downfield region between 10 and 50 ppm, and in the upfield region between 0 and -20 ppm, resolvable resonances corresponding to a small number of protons are observed. The temperature and pH behavior of these resonances have been examined. For some of the resolved resonances, the pH behavior of chemical shifts and intensities indicates that the oxidized form of the enzyme undergoes a structural transition with a pK of 5.8 +/- 0.3. On the basis of several lines of evidence, some assignments are proposed in which resolvable resonances are assigned as originating from either the heme c or the heme d1 prosthetic groups of the enzyme.     

Proton and C-13 nuclear magnetic resonance spectra of some benzoylated aldohexoses

Chemical shifts and coupling constants of ¹H-n.m.r. spectra of the perbenzoates of α-d-glucopyranose (1), β-d-glucopyranose (2), α-d-galactopyranose (3), α-d-mannopyranose (4), β-d-mannopyranose (5), and α-d-galactofuranose (6) are reported. The ¹³C-n.m.r. chemical shifts of compounds 1-3 and 6, and of penta-O-benzoyl-β-d-galactofuranose (7) are given. Mass spectra were used to differentiate the furanoses 6 and 7 from the pyranose 3.     

Proton nuclear magnetic resonance characterization of the oxidized intermediates of cytochrome c peroxidase

Oxidation of cytochrome c peroxidase with hydrogen peroxide to form the initial oxidized intermediate, cytochrome c peroxidase compound I, drastically alters the proton hyperfine nmr spectrum. In contrast to studies of horseradish peroxidase, where the spectrum of horseradish peroxidase compound I is similar to that of the native protein, cytochrome c peroxidase compound I exhibits only broad resonances near 17 and 30 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate. No unique resonances attributable to cytochrome c peroxidase compound II could be identified. These results define the molecular conditions for which resolved hyperfine resonances of the iron(IV) states of heme proteins may be observed when the data presented here are compared with the data from horseradish peroxidase. Oxidation of cytochrome c peroxidase while it is complexed to ferricytochrome c reveals that the heme resonances of cytochrome c are not influenced by the oxidation state of cytochrome c peroxidase.     

Proton nuclear magnetic resonance spectra of excised rat brain. Assignment of resonances

We have recorded 1H NMR spectra of excised rat brain at 361 MHz using two different water suppression pulse sequences. The assignment of the resonances has been carried out in perchloric acid extracts and subcellular fractions. Our results show that cytosolic proteins, membrane phospholipids and 16 different metabolites contribute to the observed spectra. The new resonances assigned allow the direct observation of myo-inositol and urea. Moreover, changes in the spectral pattern upon anesthesia, ischemic exposure of the brain and age of the rat have been recorded and correlated with the compounds producing the spectra.     

Interaction of thiocyanate with horseradish peroxidase. 1H and 15N nuclear magnetic resonance studies

Interaction of thiocyanate with horseradish peroxidase (HRP) was investigated by relaxation rate measurements (at 50.68 MHz) of the 15N resonance of thiocyanate nitrogen and by following the hyperfine shifted ring methyl proton resonances (at 500 MHz) of the heme group of SCN-.HRP solutions. At pH 4.0, the apparent dissociation constant (KD) for thiocyanate binding to HRP was deduced to be 158 mM from the relaxation rate measurements. Chemical shift changes of 1- and 8-ring methyl proton resonances in the presence of various amounts of thiocyanate at pH 4.0 yielded KD values of 166 and 136 mM, respectively. From the pH dependence of KD and the 15N resonance line width, it was observed that thiocyanate binds to HRP only under acidic conditions (pH less than 6). The binding was found to be facilitated by protonation of an acid group on the enzyme with pKa 4.0. The pH dependence of the 15N line width as well as the apparent dissociation constant were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. The KD for thiocyanate binding to HRP was also evaluated in the presence of an excess of exogenous substrates such as resorcinol, cyanide, and iodide ions. It was found that the presence of cyanide (which binds to heme iron at the sixth coordination position) and resorcinol did not have any effect on the binding of thiocyanate, indicating that the binding site of the thiocyanate ion is located away from the ferric center as well as from the aromatic donor binding site. The KD in the presence of iodide, however, showed that iodide competes with thiocyanate for binding at the same site. The distance of the bound thiocyanate ion from the ferric center was deduced from the 15N relaxation time measurements and was found to be a 6.8 A. From the distance as well as the change in the chemical shifts and line width of 1- and 8-methyl proton resonances, it is suggested that the binding site of thiocyanate may be located near heme, placed symmetrically with respect to 1- and 8-methyl groups of the heme of HRP. Similarity in the modes of binding of iodide and thiocyanate suggests that the oxidation of thiocyanate ion by H2O2 may also proceed via the two-electron transfer pathway under acidic conditions, as is the case for iodide.     

Comparison of the heme structures of horseradish peroxidase compounds X and II by resonance Raman spectroscopy

Horseradish peroxidase will catalyze the chlorination of certain substrates by sodium chlorite through an intermediate known as compound X. A chlorite-derived chlorine atom is known to be retained by compound X and has been proposed to be located at the heme active site. Although several heme structures have been proposed for compound X, including an Fe(IV)-OCl group, preliminary data previously reported by our laboratory suggested that compound X contained a heme Fe(IV) = O group, based on the similarity of a compound X resonance Raman band at 788 cm-1 to resonance Raman Fe(IV) = O stretching vibrations recently identified for horseradish peroxidase compound II and ferryl myoglobin. Isotopic studies now confirm that the 788 cm-1 resonance Raman band of compound X is, in fact, due to a heme Fe(IV) = O group, with the oxygen atom derived from chlorite. The Fe(IV) = O frequency of compound X, of horseradish peroxidase isoenzymes B and C, undergoes a pH-induced frequency shift, with behavior which appears to be the same as that previously reported for compound II, formed from the same isoenzymes. These observations strongly suggest that compounds II and X have very similar, if not identical, heme structures. The chlorine atom thus appears not to be heme-bound and may rather be located on an amino acid residue. The studies on compound X reported here were done in a pH region above pH 8, where compound X is moderately stable. The present results do not necessarily apply to compound X below pH 8.     

Proton magnetic resonance studies of peroxidases from turnip and horseradish.

Proton NMR spectra at 270 MHz have been measured for horseradish peroxidase and turnip peroxidase isoenzymes (P1, P2, P3 and P7) in both their high spin ferric native states and as the low spin ferric cyanide complexes. Resonances of amino acids near the heme have been identified and used to investigate variations in the structure of the heme crevice amongst the enzymes. Ligand proton resonances have been resolved in spectra of the cyanide complexes of the peroxidases and these provide information on the heme electronic structure. The electronic structure of the heme and the tertiary structure of the heme crevice are essentially the same in the acidic turnip isoenzymes, P1, P2 and, to a lesser extent, P3 but differ in the basic turnip enzyme, P7. The heme electronic structure and nature of the iron ligands in peroxidases are discussed. Further evidence is presented for histidine as the proximal ligand. A heme-linked ionizable group with a pK of 6.5 has been detected by NMR in the cyanide complex of horseradish peroxidase.     

Proton nuclear magnetic resonance studies of histidines in horse carbonic anhydrase I

The 250 MHz 1H-NMR spectrum of horse carbonic anhydrase I (or B) (carbonate hydro-lyase, EC 4.2.1.1) was measured as a function of pH under various conditions. Eight resonances corresponding to histidine C-2 protons and four resonances corresponding to histidine C-4 protons were identified and assigned to individual histidine residues in the enzyme molecule. Substantial similarities between horse and human carbonic anhydrases I were demonstrated. While the human enzyme has three titratable histidine residues in its active site, the horse enzyme has only two, His-67 in the human enzyme being replaced by Gln in the horse enzyme (Jabusch, J.R., Bray, R.P. and Deutsch, H.F. (1980) J. Biol. Chem. 255, 9196-9204). This substitution has small but significant effects on the behaviour of the other active-site histidines. His-64 and His-200. However, His-64 has an anomalously low pKa value also in horse isoenzyme I, as previously observed in human isoenzyme I (Campbell, I.D., Lindskog, S. and White, A.I. (1974) J. Mol. Biol. 90, 469-489).     

Transient state kinetics of the reactions of isobutyraldehyde with compounds I and II of horseradish peroxidase

Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.     

Kinetics of the oxidation of p-aminobenzoic acid catalyzed by horseradish peroxidase compounds I and II.

The kinetics of p-aminobenzoic acid oxidation catalyzed by horseradish peroxidase Compounds I and II was investigated intensively as a function of pH at 25 degrees in aqueous solutions of ionic strength 0.11. All of the rate data were collected from single turnover experiments involving reactions of a single enzyme compound. In reactions of both compounds, deviations from first order behavior with respect to the enzyme were observed at high pH values which were explained in terms of a free radical interaction of product with the enzyme. The effect could be eliminated with sufficient excess of substrate. Kinetic behavior which deviated from first order in substrate, observed at low pH, was explained by a mechanism involving an enzyme-substrate complex which reacted with an additional molecule of substrate but at a slower rate. The pH dependence of the second order rate constants for the reaction of p-aminobenzoic acid with free Compounds I and II is similar to results obtained for the comparable reactions of ferrocyanide, suggesting similar proton-transfer mechanisms for both reducing substrates. The reduction of Compound II by p-aminobenzoic acid appeared to be influenced by two ionizable groups on the enzyme which affect the electronic environment of the heme. The lack of influence of substrate ionizable groups on the rate of the Compound II reaction indicated that potential differences in reactivities of NH2C6H4COO- and NH2C6H4COOH were levelled by the diffusion-controlled limit in the acid region of pH. The reduction of Compound I by p-aminobenzoic acid was not diffusion-controlled and the rate-pH profile could be explained in terms of three acid ionizations, two on the substrate and one on Compound I.     

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 degrees C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I-NADH reaction can be explained in terms of a single ionization of pKa = 4.7 +/- 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-II-NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 +/- 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II-NADH reaction was observed. Over the pH range of 4-10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 X 10(5) to 5.6 X 10(2) M-1 s-1 and of HRP-II with NADH varied from 4.4 X 10(4) to 4.1 M-1 s-1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.     

Proton nuclear magnetic resonance study of cobrotoxin.

Cobrotoxin (Mr 6949), which binds tightly to the acetylcholine receptors, contains no phenylalanines and only two histidines, two tyrosines, and one tryptophan that result in well-resolved aromatic proton resonances in D2O at 360 MHz. His-32, Tyr-25, and the Trp are essential for toxicity and may interact with the acetylcholine receptor. We assign two titratable resonances (pKa = 5.1) at delta = 9.0 and 7.5 ppm at pH 2.5 and at 7.7 and 7.1 ppm at pH 9.5 to the C-2 and C-4 ring protons, respectively, of His-4. Two other titratable resonances (pKa = 5.7) at delta = 8.8 and 6.9 ppm at pH 2.5 and at 7.8 and 6.7 ppm at pH 9.5 are assigned to the C-2 and C-4 ring protons of His-32, respectively. The differences in delta values of the two histidines reflect chemically different microenvironments while their low pKa values could arise from nearby positive charges. A methyl resonance gradually shifts upfield to delta approximately 0.4 ppm as His-4 is deprotonated and is tentatively assigned to the methyl group of Thr-14 or Thr-15 which, from published X-ray studies of neurotoxins, are located in the vicinity of His-4. Further, we have identified the aromatic resonances of the invariant tryptophan and individual tyrosines and the methyl resonance of one of the two isoleucines in the molecule. Several broad nontitrating resonances of labile protons which disappear at pH greater than 9 may arise from amide groups of the beta sheet in cobrotoxin.