In vitro formation of a c -type cytochrome

Abstract

C-type cytochromes are essential for almost all organisms; they are characterized by the covalent attachment of heme to protein through two thioether bonds to a Cys-Xaa-Xaa-Cys-His peptide motif. Here we show, contrary to opinion of 30 years standing, that a c-type cytochrome can form from heme and apoprotein in vitro under mild conditions and in the absence of any biosynthesis apparatus. This reaction occurs provided formation of a disulfide bond within the Cys-Xaa-Xaa-Cys-His motif is avoided. There are important implications for understanding in vivo cytochrome c assembly.

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

“…1 and the protein was fully reduced. The presence of both DTT and dithionite has been used in other work with cytochrome c (see, for example, [8,14,15]) in which we have observed that DTT is needed to maintain cysteine thiol group integrity and dithionite is needed to maintain the ferrous state of the haem. The UV-visible spectrum of the solution was taken every 30 min for 18 h, at which point the reaction was deemed to be complete.…”

“…The latter result is significant because thioether bond formation might, in principle, be achieved via a radical mechanism that depends upon the availability of an oxidant [22]. Although several lines of in vitro [8] and in vivo [23] evidence have suggested that reducing conditions promote thioether bond formation as seen in c-type cytochromes, these observations might relate to a requirement for ferrous haem and/or prevention of the two cysteine residues of the CXXCH motif becoming a disulfide, rather than a requirement for the bond formation itself. The fact that oxidative conditions (i.e.…”

“…Thus Barker et al [22] proposed a radical reaction involving consumption of molecular oxygen, to account for some of the products observed when a variant of cytochrome b 5 , bearing a cysteine residue in the haem-binding site, was expressed in the E. coli cytoplasm. Daltrop et al [8] reported that reaction of apocytochrome c 552 with ferric haem in vitro led to undefined mixed products. To what extent these non-physiological products depend upon complex factors such as the tendency of thiol groups (either of the protein or of dithiothreitol) to reduce ferric haem bound to different proteins, and thus with different reduction potentials, is not known.…”

“…Similarly, the spectrum of the reduced pyridine haemochromagen derivative now had an α-band that had shifted from 556 to 553.5 nm ( Figure 2B). These spectral changes are diagnostic of the covalent attachment to one of the two vinyl groups on the haem [8]. Reactions at higher pH (8.4), which would have allowed assessment of the importance or otherwise of the (deprotonated) thiolate form of the cysteine side chain, were not accessible as a result of protein precipitation over the timescale of the reaction.…”

“…First, it proved possible to prepare an apo form of a mono haem c-type cytochrome, from a thermophilic bacterium and named cytochrome c 552 , that had intact thiol groups in the CXXCH motif and which would adopt a near-native three-dimensional fold on non-covalent binding of haem. Incubation of the non-covalent haem-apocytochrome complex under reducing conditions resulted in the formation in vitro of material that was spectroscopically indistinguishable from the native c-type cytochrome [8]. It is believed that a close juxtaposition of the thiol groups to the vinyl groups of the haem promoted the formation of covalent bonds through attack of the thiols on the vinyl groups, which are not especially activated for either electrophilic or nucleophilic attack, of the ferrous haem.…”