title: A suite of de novo c-type cytochromes for functional oxidoreductase engineering.
abstract: Central to the design of an efficient de novo enzyme is a robust yet mutable protein scaffold. The maquette approach to protein design offers precisely this, employing simple four-α-helix bundle scaffolds devoid of evolutionary complexity and with proven tolerance towards iterative protein engineering. We recently described the design of C2, a de novo designed c-type cytochrome maquette that undergoes post-translational modification in E. coli to covalently graft heme onto the protein backbone in vivo. This de novo cytochrome is capable of reversible oxygen binding, an obligate step in the catalytic cycle of many oxygen-activating oxidoreductases. Here we demonstrate the flexibility of both the maquette platform and the post-translational machinery of E. coli by creating a suite of functional de novo designed c-type cytochromes. We explore the engineering tolerances of the maquette by selecting alternative binding sites for heme C attachment and creating di-heme maquettes either by appending an additional heme C binding motif to the maquette scaffold or by binding heme B through simple bis-histidine ligation to a second binding site. The new designs retain the essential properties of the parent design but with significant improvements in structural stability. Molecular dynamics simulations aid the rationalization of these functional improvements while providing insight into the rules for engineering heme C binding sites in future iterations. This versatile, functional suite of de novo c-type cytochromes shows significant promise in providing robust platforms for the future engineering of de novo oxygen-activating oxidoreductases. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electron transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Watkins
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Craig T
authors.initials: CT
authors.lastname: Armstrong
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK; School of Chemistry, University of Bristol, Bristol BS8 1TS, UK.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Beesley
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Marsh
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Jonathan M X
authors.initials: JMX
authors.lastname: Jenkins
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Sessions
authors.affiliation: School of Chemistry, University of Bristol, Bristol BS8 1TS, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Mann
authors.affiliation: School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK. Electronic address: ross.anderson@bristol.ac.uk.
authors.forename: J L
authors.initials: JL
authors.lastname: Ross Anderson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D003574:Cytochrome c Group; D011993:Recombinant Fusion Proteins; C031690:heme C; D006418:Heme; D010088:Oxidoreductases
keywords: Cytochrome c; E. coli cytochrome c biogenesis system I; Enzyme design; Four-helix bundle; Heme C; Maquette; Oxygen activation; Oxygen binding; Protein design