Structural studies by proton‐NMR spectroscopy of plant horseradish peroxidase C, the wild‐type recombinant protein from Escherichia coli and two protein variants, Phe41 → Val and Arg38 → Lys

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Abstract
Wild-type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41 → Val anfd Arg38 → Lys, have been characterised using both one- and two-dimensional NMR spectroscopy. Proton NMR spectra recorded in both resting and cyanide-ligated states of the proteins were compared with those of the corresponding plant peroxidase. The latter contains 18% carbohydrate in eight N-linked oligosaccharide side chains whereas the recombinant proteins are expressed in non-glycosylated form. The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydreate-linked resonances in the former indicating that their solution structures are highly similar. This comparison also identifies classes of carbohydrate resonances in the plant enzymes which provides new information on the local environment and mobility of the oligosaccharide side chains. Comparison of the spectra of the Cyanide-ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem-linked resonances. These could not be rationalised simply on the basis of the local perturbation expected from a single-site substitution. The two substititions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group. Inspection of the spectra of the Phe41 → Val varient also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly made for the Phe41 → Val varient were also a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid.

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