PubMed · 6363473
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N S Penneys. 1983. Immunoperoxidase methods in skin pathology.. https://doi.org/10.1111/j.1600-0560.1983.tb01496.x
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The active species, Compound I, of horseradish peroxidase (HRP) has been investigated by quantum mechanical/molecular mechanical (QM/MM) calculations using 10 different QM regions. In accord with experimental data, the lowest doublet and quartet states are found to be virtually degenerate, with two unpaired electrons on the FeO moiety and one localized on the porphyrin in an a(2u)-dominant orbital with a minor, but nonnegligible, a(1u) component. The proximal ligand appears to be imidazole rather than imidazolate. The hydrogen-bonding network around the FeO moiety (i.e., Arg38 and His42) has significant influence on the axial bonds and the spin density distribution in the FeO moiety. Including this network in the QM region was found to be essential for reproducing the experimental Mössbauer parameters. The protein environment shapes most of the subtle features of Compound I of HRP.
The oxidative polymerization of 1-naphthol was investigated in the presence of horseradish peroxidase (HRP). Naphthol polymerization products (NPP) were characterized for their relative polarity using octanol--water partitioning experiments and reverse-phase high pressure liquid chromatography, for structure using size exclusion chromatography and liquid chromatography-mass spectrometry (LC/MS), and for ecotoxicity using inhibition of bacterial bioluminescence. Peroxidase addition resulted in the production of soluble and insoluble NPP. Soluble NPP was predominantly more polar than the parent naphthol and comprised of trimers and tetramers. Insoluble NPP oligomers included dimers, trimers and tetramers. The net aqueous-phase toxicity was significantly reduced due to polymer formation and subsequent precipitation. A reaction model deduced from the LC/MS fragmentation patterns of trimeric naphthol was proposed for NPP formation. Results from this study suggest that HRP-mediated treatment of naphthol contaminated soils can achieve risk reduction through (i) the formation of large hydrophobic oligomers that are immobilized on the soil matrix; and (ii) reduction in aqueous-phase toxicity due to polymer precipitation.
Studies on horseradish peroxidase C and other haem peroxidases have been carried out on selected mutants in the distal haem cavity providing insight into the functional importance of the distal residues. Recent work has demonstrated that proximal structural features can also exert an important influence in determining the electronic structure of the haem pocket. To extend our understanding of the significance of proximal characteristics in regulating haem properties the proximal Thr171Ser mutant has been constructed. Thr171 is an important linking residue between the structural proximal Ca2+ ion and the proximal haem ligand, in particular the methyl group of Thr171 interdigitates with other proximal residues in the core of the enzyme. Although the mutation induces no significant changes to the functional properties of the enzyme, electronic absorption and resonance Raman spectroscopy reveal that it has a highly selective affect on the reduced state of the enzyme, effectively stabilizing it, whilst the electronic properties of the Fe(III) state unchanged and essentially identical to those of the native protein. This results in a significant change in the Fe2+/Fe3+ redox potential of the mutant. It is concluded that the unusual properties of the Thr171Ser mutant reflect the loss of a structural restraint in the proximal haem pocket that allows 'slippage' of the proximal haem ligand, but only in the reduced state. This is a remarkably subtle and specific effect that appears to increase the flexibility of the reduced state of the mutant compared to that of the wild-type protein.