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Conformational transitions and redox potential shifts of cytochrome P450 induced by immobilization

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Abstract

Cytochrome P450 (P450) from Pseudomonas putida was immobilized on Ag electrodes coated with self-assembled monolayers (SAMs) via electrostatic and hydrophobic interactions as well as by covalent cross-linking. The redox and conformational equilibria of the immobilized protein were studied by potential-dependent surface-enhanced resonance Raman spectroscopy. All immobilization conditions lead to the formation of the cytochrome P420 (P420) form of the enzyme. The redox potential of the electrostatically adsorbed P420 is significantly more positive than in solution and shows a steady downshift upon shortening of the length of the carboxyl-terminated SAMs, i.e., upon increasing the strength of the local electric field. Thus, two opposing effects modulate the redox potential of the adsorbed enzyme. First, the increased hydrophobicity of the heme environment brought about by immobilization on the SAM tends to upshift the redox potential by stabilizing the formally neutral ferrous form. Second, increasing electric fields tend to stabilize the positively charged ferric form, producing the opposite effect. The results provide insight into the parameters that control the structure and redox properties of heme proteins and contribute to the understanding of the apparently anomalous behavior of P450 enzymes in bioelectronic devices.

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Acknowledgements

Financial support by the FCT (POCTI-BIO−43105-2001) and the DFG (Sfb498-A8 and Ju229/4-3) is gratefully acknowledged.

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Authors and Affiliations

  1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal

    Smilja Todorovic, Peter Hildebrandt & Daniel H. Murgida

  2. Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, 13125, Berlin, Germany

    Christiane Jung

  3. Institut für Chemie, Max-Volmer-Laboratorium für Biophysikalische Chemie, Sekr. PC14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623, Berlin, Germany

    Peter Hildebrandt & Daniel H. Murgida

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  1. Smilja Todorovic
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  2. Christiane Jung
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  4. Daniel H. Murgida
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Correspondence to Daniel H. Murgida.

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Todorovic, S., Jung, C., Hildebrandt, P. et al. Conformational transitions and redox potential shifts of cytochrome P450 induced by immobilization. J Biol Inorg Chem 11, 119–127 (2006). https://doi.org/10.1007/s00775-005-0054-9

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  • DOI: https://doi.org/10.1007/s00775-005-0054-9

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