Cytochrome P450 from photobacterium profundum SS9, a piezophilic bacterium, exhibits a tightened control of water access to the active site

Elena V. Sineva, Dmitri R. Davydov

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

We report cloning, expression in Escherichia coli, and purification of cytochrome P450 from a deep-sea bacterium Photobacterium profundum strain SS9 (P450-SS9). The enzyme, which is predominately high spin (86%) in the absence of any added ligand, binds fatty acids and their derivatives and exhibits the highest affinity for myristic acid. Binding of the majority of saturated fatty acids displaces the spin equilibrium further toward the high-spin state, whereas the interactions with unsaturated fatty acids and their derivatives (arachidonoylglycine) have the opposite effect. Pressure perturbation studies showed that increasing pressure fails to displace the spin equilibrium completely to the low-spin state in the ligand-free P450-SS9 or in the complexes with either myristic acid or arachidonoylglycine. Stabilization of high-spin P450-SS9 signifies a pressure-induced transition to a state with reduced accessibility of the active site. This transition, which is apparently associated with substantial hydration of the protein, is characterized by the reaction volume change (δV) around -100 to -200 mL/mol and P1/2 of 300-800 bar, which is close to the pressure of habitation of P. profundum. The transition to a state with confined water accessibility is hypothesized to represent a common feature of cytochromes P450 that serves to coordinate heme pocket hydration with ligand binding and the redox state. Displacement of the conformational equilibrium toward the "closed" state in P450-SS9 (even ligand-free) may have evolved to allow the protein to adapt to enhanced protein hydration at high hydrostatic pressures.

Original languageEnglish (US)
Pages (from-to)10636-10646
Number of pages11
JournalBiochemistry
Volume49
Issue number50
DOIs
StatePublished - Dec 21 2010

All Science Journal Classification (ASJC) codes

  • Biochemistry

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