Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette

J. L. Ross Anderson*, Craig T. Armstrong, Goutham Kodali, Bruce R. Lichtenstein, Daniel W. Watkins, Joshua A. Mancini, Aimee L. Boyle, Tammer A. Farid, Matthew P. Crump, Christopher C. Moser, P. Leslie Dutton

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo.

Original languageEnglish
Pages (from-to)507-514
Number of pages8
JournalChemical Science
Issue number2
Early online date31 Oct 2013
Publication statusPublished - 1 Feb 2014


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