In Vitro Studies on Thioether Bond Formation between Hydrogenobacter thermophilus Apocytochrome c552 with Metalloprotoporphyrin Derivatives

CytochromecBiogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

“…Again, the reactions require a reduced heme, using dithionite. Daltrop et al have analyzed a unique cytochrome c from a thermophile, which, unlike most cytochromes c, folds properly even when the heme is not attached (39)(40)(41). They have made some important observations concerning the spontaneous nature of the thioether ligations, which again occur only when the iron is reduced (to Fe 2ϩ ).…”

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].…”

“…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.…”

“…With an emerging understanding, albeit not complete, of the diverse cross-links in natural heme proteins, most of these cross-links have been successfully recreated in natural or de novo proteins, such as Cys-heme [24,26,27,33,[41][42][43][44][45], Met-heme [53], His-heme [70][71][72], Trp/Tyr-heme [78] and Glu/Asp-heme [82,106,[108][109][110][111] cross-links. These achievements, in turn, have enhanced our knowledge of how heme-protein cross-link regulates the structure and function of heme proteins in nature.…”

“…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.…”