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Trapping of peptide-based surrogates in an artificially created channel of cytochrome c peroxidase

¹ Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA
² Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA

Reprint requests to: David B. Goodin, Department of Molecular Biology, MB8, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: dbg{at}scripps.edu; fax: (858) 784-2857.

As recently described, the deliberate removal of the proposed electron transfer pathway from cytochrome c peroxidase resulted in the formation of an extended ligand-binding channel. The engineered channel formed a template for the removed peptide segment, suggesting that synthetic surrogates might be introduced to replace the native electron transfer pathway. This approach could be united with the recent development of sensitizer-linked substrates to initiate and study electron transfer, allowing access to unresolved issues about redox mechanism of the enzyme. Here, we present the design, synthesis, and screening of a peptide library containing natural and unnatural amino acids to identify the structural determinants for binding this channel mutant. Only one peptide, (benzimidazole-propionic acid)-Gly-Ala-Ala, appeared to interact, and gave evidence for both reversible and kinetically trapped binding, suggesting multiple conformations for the channel protein. Notably, this peptide was the most analogous to the removed electron transfer sequence, supporting the use of a cavity-template strategy for design of specific sensitizer-linked substrates as replacements for the native electron transfer pathway.

Keywords: Heme enzyme; protein engineering; protein cavities; cavity complementation; chemical rescue; electron transfer pathway; sensitizer-linked substrates

Abbreviations: CCP, cytochrome c peroxidase • SLS, sensitizer-linked substrate • cyt c, cytochrome c • ET, electron transfer • P450cam, cytochrome P450cam • HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate • HOBt, N-hydroxybenzotriazole • Fmoc, 9-fluroenyl-methoxycarybonyl • Dde, 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene-ethane • DMF, N,N-dimethylformamide • TEA, triethylamine • TFA, trifluoroacetic acid • TIS, tri-isopropylsilane • BTP, bistrispropane • CA, citric acid • ITC, isothermal titration calorimetry • GuHCl, guanidine hydrochloride • Ru-C₁₁-Im, Ru-C₉-EB, Ru-F₈bp-Im, Ru-F₉bp (Ru = [Ru^II(bpy)₃²+, C₁₁ = (CH₂)₁₁—, C₉ = —(CH₂)₉—, Im = imidazole, EB = ethylbenzene, F₈bp = 4,4'-octafluorobiphenyl, F₉bp = 4,4'-nonafluorobiphenyl)

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