Previous work [Metcalfe, Ott, Patel, Singh, Mistry, Goff and Raven (2004) J. Am. Chem. Soc. 126, 16242–16248] has shown that the introduction of a methionine residue (S160M variant) close to the 2-vinyl group of the haem in ascorbate peroxidase leads to the formation of a covalent haem–methionine linkage under oxidative conditions (i.e. on reaction with H2O2). In the present study, spectroscopic, HPLC and mass spectrometric evidence is presented to show that covalent attachment of the haem to an engineered cysteine residue can also occur in the S160C variant, but, in this case, under reducing conditions analogous to those used in the formation of covalent links in cytochrome c. The data add an extra dimension to our understanding of haem to protein covalent bond formation because they show that different types of covalent attachment (one requiring an oxidative mechanism, the other a reductive pathway) are both accessible within same protein architecture.
Tuning the formation of a covalent haem–protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase
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Clive L. Metcalfe, Oliver Daltrop, Stuart J. Ferguson, Emma Lloyd Raven; Tuning the formation of a covalent haem–protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase. Biochem J 15 December 2007; 408 (3): 355–361. doi: https://doi.org/10.1042/BJ20071041
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