Constrained water access to the active site of cytochrome P450 from the piezophilic bacterium Photobacterium profundum

Abstract

Living species inhabiting ocean deeps must adapt to high hydrostatic pressure. This adaptation, which must enable functioning under conditions of promoted protein hydration, is especially important for proteins such as cytochromes P450 that exhibit functionally important hydration–dehydration dynamics. Here we study the interactions of substrates with cytochrome P450-SS9, a putative fatty acid hydroxylase from the piezophilic bacterium Photobacterium profundum SS9, and characterize the protein's barotropic properties. Comparison of P450-SS9 with cytochrome P450BM-3, a mesophilic fatty acid hydroxylase, suggests that P450-SS9 is characterized by severely confined accessibility and low water occupancy of the active site. This feature may reveal a mechanism for the structural adaptation of the piezophilic enzyme. We also demonstrate that saturated and unsaturated fatty acids exert opposite effects on solvent accessibility and hydration of the active site. Modulation of the protein conformation by fatty acids is hypothesized to have an important physiological function in the piezophile.

Keywords:

• pressure-perturbation spectroscopy
• cytochrome P450
• piezophilic organism
• protein hydration
• substrate binding

Acknowledgements

The authors are grateful to Dr James R. Halpert (Skaggs School of Pharmacy and Pharmaceutical Science, UC San Diego) for his continuous support and valuable comments and to Dr Douglas H. Bartlett (Scripps Institution of Oceanography, UC San Diego) for providing us with DNA of P. profundum SS9. We highly appreciate a kind gift of the purified heme domain of cytochrome P450BM-3 provided by Dr Donovan C. Haines (Sam Houston State University, Huntsville, TX, USA). This research was supported by NIH grant GM054995.