Tuning the formation of a covalent haem–protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase

Abstract

Raven (2004) J. Am. Chem. Soc. 126, [16242][16243][16244][16245][16246][16247][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 H 2 O 2 ). 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.

The broad diversity of heme-protein cross-links: An overview

“…The formation of double His-heme cross-links in L79H PCC 6803 under reducing conditions in vitro is of particular interest, as it parallels the double Cys-heme cross-links in the majority of cyts c. Moreover, even when the native cross-link of His117-N ε -2-vinyl-C α was removed, the double mutant of L79H/H117A PCC 6803 can form a single cross-link of His79-N ε -4-vinyl-C α . These observations indicate that each of the double His-heme cross-links form independently in cyanobacteria Hbs, a behavior similar to the double Cys-heme cross-links in cyts c [41][42][43][44][45].…”

“…Moreover, the thioether bond can form with heme derivatives containing other metals such as Zn 2+ or Mn 2+ [42,43], which provides a convenient strategy for design of artificial metalloproteins containing a non-natural prosthetic group. In agreement with these findings, Metcalfe et al [44] showed that introduction of a Cys (S160C mutation) close to the heme 2-vinyl group in recombinant pea cytosolic ascorbate peroxidase (APX) leads to the formation of a thioether bond, during reconstitution of the apo-protein with heme in vitro under reducing conditions. In addition to the native protein scaffolds of cyt c 552 [41][42][43] and APX [44], thioether bond could also be generated in de novo four-helix bundle proteins by reacting with heme in vitro [45], which opens a way to design functional de novo heme proteins with a non-dissociable heme cofactor.…”

“…To provide mechanistic information and to duplicate the sulfonium ion bond in MPO, Raven and co-workers [53] engineered a methionine residue (S160M mutation) close to the 2-vinyl group in APX at the same position for construction of a thioether bond [44]. Although the S160M APX mutant was isolated as an apo-protein, reconstitution of it with heme followed by exposure to H 2 O 2 was found to form a sulfonium ion bond between Met160 and the vinyl group, with an addition of hydroxyl group at the C α of vinyl group by mass spectrometric analysis.…”

“…Based on kinetic studies that identified a reaction intermediate, oxoferryl heme π-cation radical (Compound I), the authors proposed an autocatalytic mechanism for formation of the sulfonium ion bond (Scheme 2) [53]. It is interesting to observe that the formation of sulfonium ion bond and thioether bond was under oxidative and reducing conditions, respectively, whereas each of Met-heme and Cys-heme cross-links could be formed within the same protein scaffold of APX [44]. It should be noted that although the construction of a sulfonium ion bond in APX was succeeded, attempts to construct the bond in other peroxidases such as LPO [54] and horseradish peroxidase (HRP) [55] were unsuccessful.…”

“…APX is an ideal protein for elucidating the mechanism of hemeprotein cross-links, and a heme-protein cross-link can be constructed by introducing Cys or Met close to the heme vinyl group [44,53]. Additionally, as discovered by Raven and co-workers [75], a hemeprotein cross-link could also be formed in native APX, when the protein was exposed to H 2 O 2 under noncatalytic conditions (i.e., in the absence of substrate) for a long time (~8 h).…”