title: Engineered holocytochromecsynthases that biosynthesize new cytochromesc.
abstract: Cytochromec(cytc), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochromecsynthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cytcIn cytc, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochromecfrom HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cytcin recombinantEscherichia coliFocusing on HCCS E159A, novel cytcvariants in quantities that are sufficient for biophysical analysis are biosynthesized. CytcH19M, the first bis-Met liganded cytc, is compared with other axial ligand variants (M81A, M81H) and single thioether cytcvariants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; -19 mV, M81A; -69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cytcfrom the HCCS active site; thus, we term these "release mutants."
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, MO 63130.
authors.forename: Deanna L
authors.initials: DL
authors.lastname: Mendez
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, MO 63130.
authors.forename: Shalon E
authors.initials: SE
authors.lastname: Babbitt
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, MO 63130.
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: King
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, MO 63130.
authors.forename: John
authors.initials: J
authors.lastname: D'Alessandro
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Watson
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, MO 63130.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Blankenship
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130.
authors.forename: Liviu M
authors.initials: LM
authors.lastname: Mirica
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, MO 63130; kranz@wustl.edu.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Kranz
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D045304:Cytochromes c / Q000096:biosynthesis* / Q000235:genetics; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008190:Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D006418:Heme; D045304:Cytochromes c; D008190:Lyases; C031581:cytochrome C synthetase
keywords: HCCS; axial ligands; cytochrome c; heme; spectroscopy