Design and Synthesis of de Novo Cytochromes c

Abstract

Natural c-type cytochromes are characterized by the consensus Cys-X-X-Cys-His heme-binding motif (where X is any amino acid) by which the heme is covalently attached to protein by the addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of the heme. In this work, the consensus sequence was used for the heme-binding site of a designed four-helix bundle, and the apoproteins with either a histidine residue or a methionine residue positioned at the sixth coordination site were synthesized and reacted with iron protoporphyrin IX (protoheme) under mild reducing conditions in vitro. These polypeptides bound one heme per helix-loop-helix monomer via a single thioether bond and formed four-helix bundle dimers in the holo forms as designed. They exhibited visible absorption spectra characteristic of c-type cytochromes, in which the absorption bands shifted to lower wavelengths in comparison with the b-type heme binding intermediates of the same proteins. Unexpectedly, the designed cytochromes c with bis-His-coordinated heme iron exhibited oxidation-reduction potentials similar to those of their b-type intermediates, which have no thioether bond. Furthermore, the cytochrome c with His and Met residues as the axial ligands exhibited redox potentials increased by only 15-30 mV in comparison with the cytochrome with the bis-His coordination. These results indicate that highly positive redox potentials of natural cytochromes c are not only due to the heme covalent structure, including the Met ligation, but also due to noncovalent and hydrophobic environments surrounding the heme. The covalent attachment of heme to the polypeptide in natural cytochromes c may contribute to their higher redox potentials by reducing the thermodynamic stability of the oxidized forms relatively against that of the reduced forms without the loss of heme.

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

“…Nevertheless, under reductive conditions, thioether bond formation might prove to be general. Thus the newly discovered single thioether bond between haem and polypeptide in the cytochrome b 6 f complex [28,29] could prove to be an additional in vivo example, while thioether bond formation has been seen recently following in vitro reaction of designer fourhelix bundle proteins and haem [30]. Thus suitable mutagenesis, followed by either oxidizing or reducing reaction conditions as appropriate, may provide routes to a variety of engineered covalent links between polypeptide and haem.…”

Five Amino Acid Residues Responsible for the High Stability of Hydrogenobacter thermophilus Cytochrome c552

“…However, there is still a necessity to understand the general principle of protein stabilization, to study a strategy employed in an individual protein. Further accumulation of examples of reciprocal residue swapping between homologous native proteins together with artificially designed proteins (21,22) will rationally reveal the principle of protein stability.…”

Molecular modeling of cytochrome b 5 with a single cytochrome c-like thioether linkage

“…A previous study showed that c-type R98C cyt b 562 has a very similar solution structure to that of the b-type cyt b 562 [15]. In addition, during the formation of cyt c species in vitro, the b-type intermediates were found to have similar spectroscopic properties to those of the c-type products [54].…”

“…Additionally, the spontaneous formation of a single thioether bond can occur in vitro, as apo-cyt c species contain either the AXXCH or CXXAH motif [53]. This method has also been successfully applied to the design and synthesis of de novo cyt c species [54]. On the other hand, b-type cytochromes can be converted into c-type cytochromes, as exemplified by cyt b 5 and cyt b 562 [12][13][14][15][16].…”