Identification of the catalytically important amino acid residue resonances in ferric low-spin horseradish peroxidase with nuclear Overhauser effect measurements

Thanabal, V.; Ropp, Jeffrey S. De; Mar, Gerd N. La

doi:10.1021/ja00258a043

Cited by 121 publications

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“…No cross-peaks were observed between either of the C7 and C18 haem methyls and aromatic proton resonances, in contrast to cyanide-ligated HRP C (Thanabal et al, 1987;Veitch and Williams, 1990). Similarly, the characteristic NOE cross-peak between haem methyl ClSH, and an upfield-shifted methyl was absent.…”

Section: Amino Acid Proton Resonance Assignmentsmentioning

confidence: 77%

NMR studies of recombinant Coprinus peroxidase and three site‐directed mutants

Veitch

Tams

Vind

et al. 1994

European Journal of Biochemistry

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Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 andlor Asn157 was replaced by Phe. Analysis of one-and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP.The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phel56 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidasesThe frequency with which peroxidase enzymes are being isolated and characterised from new sources far outweighs progress in our knowledge both of their three-dimensional structures and of their interactions with substrate and ligand molecules. A peroxidase in this category is that recently isolated and characterised from the ink cap basidiomycete Co-

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Section: Amino Acid Proton Resonance Assignmentsmentioning

confidence: 77%

NMR studies of recombinant Coprinus peroxidase and three site‐directed mutants

Veitch

Tams

Vind

et al. 1994

European Journal of Biochemistry

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“…This protein form, although not physiologically active, has been the most favorable system on which paramagnetic NMR investigations are carried out (35,37,70,71). The short electronic relaxation times and large magnetic anisotropy of the low spin peroxidase cyanide complexes give much sharper and better resolved signals in their proton NMR spectra, providing much more information about the electronic, magnetic, and molecular structural properties of the heme pocket as compared with the native, high spin resting forms (27,38,50,72). In addition, the cyanide adduct of heme peroxidases has been implicated as an important analogue for the active, oxidized low spin enzyme intermediates for which proton NMR spectroscopy is currently inapplicable due to the large resonance line widths (73).…”

Section: Resultsmentioning

confidence: 99%

“…The predicted distances of these signals to the heme iron center is about 4.5 Å (Table II) inequivalent protons in noncoordinating groups and their distances to the paramagnetic center (50), assuming that heme methyl protons have an average distance of 6.1 Å from the iron. Therefore, it is reasonable, in the absence of the crystal coordinates of CPO, to assign these signals to the heme mesoprotons, since they are the only remaining protons close enough to heme iron to experience such sufficient paramagnetic relaxation (54).…”

Section: Resonance Assignmentmentioning

confidence: 99%

“…Compared with the extensive and in depth NMR studies on CcP (28 -36) and horseradish peroxidase (34,(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53), relatively few NMR studies of CPO have been reported (54 -58). Furthermore, the most powerful NMR approach, the two-dimensional NMR technique that has led to the unambiguous assignment of major hyperfine-shifted signals in a number of heme peroxidases (59), has not been applied to the investigation of CPO.…”

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confidence: 99%

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Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase

Wang

Tachikawa

et al. 2003

Journal of Biological Chemistry

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“…'H-NMR relaxation measurements performed by Sakurada et al 1191 place the aromatic protons of a range of donor molecules (including 2-methoxy-4-methyl phenol, resorcinol and benzhydroxamic acid) between 0.84 nm and 1.2 nm from the haem iron. In addition, Veitch et al [15,161 and La Mar et al have, by means of measurement of NOE connectivities, implicated the active-site amino acids His42, two Phe residues (designated PheA and PheB ; PheA was tentatively assigned to Phe142) and an isoleucine residue (IleX was initially believed to be Leu237 [20] and later corrected to Ile244 on the basis of a more accurate sequence alignment [23] Both direct and indirect techniques have led to the consensus opinion that in HRP-C the aromatic-donor-binding site positions the substrate in a flexible hydrophobic pocket close and distal to the haem edge, in the vicinity of the haem C18-methyl 14,16,191. Here we report a series of studies in which we have quantified the covalent-haem-modification pattern obtained during the phenylhydrazine inactivation of a range of HRP-C* mutants which have either been implicated as structural components of the aromatic-donor-binding site (Phe142 and Phe143) or are catalytically important (Arg38 and His42; Fig.…”

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confidence: 99%

Probing the Aromatic‐Donor‐Binding Site of Horseradish Peroxidase Using Site‐Directed Mutagenesis and the Suicide Substrate Phenylhydrazine

Gilfoyle

Rodrı́guez-López

Smith

1996

European Journal of Biochemistry

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The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-accesschannel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (8-meso phenyl) and C1 8-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03 % of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkably constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why phenylhydrazine-treated C. cinereus peroxidase was more resistant to haem modification and phenyl-radical-based inactivation than HRP-C.Keywords: horseradish peroxidase; suicide inactivation; phenylhydrazine; site-directed mutagenesis.Classical peroxidases, including horseradish peroxidase (HRP), have been shown to catalyse the transfer of electrons in two distinct one-electron steps from a wide range of aromatic donor molecules [l]. The reaction, initiated by hydrogen peroxide, provides two oxidising equivalents to the resting haem prosthetic group and yields compound I, an oxy-ferryl-[Fe(IV) = 01-based porphyrin radical cation. Although compounds I and 11, the high oxidation state intermediates formed during the catalytic cycle, have been well described [2], continued efforts to understand structure/function relationships in HRP have been hampered by the lack of a high-resolution structure of a class 111 (higher plant) peroxidase. Despite the wealth of biocheniical data for this archetypal peroxidase it has been necessary to rely

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Identification of the catalytically important amino acid residue resonances in ferric low-spin horseradish peroxidase with nuclear Overhauser effect measurements

Cited by 121 publications

References 0 publications

NMR studies of recombinant Coprinus peroxidase and three site‐directed mutants

NMR studies of recombinant Coprinus peroxidase and three site‐directed mutants

Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase

Probing the Aromatic‐Donor‐Binding Site of Horseradish Peroxidase Using Site‐Directed Mutagenesis and the Suicide Substrate Phenylhydrazine

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