We have explored the adaptation of the cytochromes P450 (P450) of deep-sea bacteria to high hydrostatic pressures. Strict conservation of the protein fold and functional importance of proteinbound water make P450 a unique subject for the studies of high pressure adaptation. Earlier we expressed and purified a fatty-acid binding P450 from the deep-sea bacteria Photobacterium profundum SS9 (CYP261C1). Here we report purification and initial characterization of its mesophilic ortholog from the shallow-water P. profundum 3TCK (CYP261C2), as well as another piezophilic enzyme, CYP261D1 from deep-see Moritella sp. PE36. Comparison of the three enzymes revealed a striking peculiarity of the piezophilic enzymes. Both CYP261C1 and CYP261D1 possess an apparent pressure-induced conformational toggle actuated at the pressures commensurate with the physiological pressure of habitation of the host bacteria. Furthermore, in contrast to CYP261C2, the piezophilic CYP261 enzymes may be chromatographically separated into two fractions with different properties, and different thermodynamic parameters of spin equilibrium in particular. According to our concept, the changes in the energy landscape that evolved in pressure-tolerant enzymes must stabilize the less-hydrated, closed conformers, which may be transient in the catalytic mechanisms of non-piezophilic enzymes. The studies of enzymes of piezophiles should help unravel the mechanisms that control water access during the catalytic cycle.Keywords pressure-tolerant enzymes; piezophiles; conformational heterogeneity; cytochrome; P450; monooxygenases; molecular mechanism Cytochromes P450, the heme-thiolate enzymes that catalyze oxidation of a wide variety of endogenous and exogenous lipophilic compounds, play diverse physiological functions varying from the synthesis of steroid hormones, prostaglandins and second messengers to oxidation of exogenous compounds, such as drugs and other xenobiotics. Monooxygenation reactions require the input of two electrons (supplied by flavoprotein or ferredoxin partners) used to activate the dioxygen molecule. One oxygen atom is then inserted into the substrate, while the second atom is reduced and released in the form of a water molecule. However, an important fraction of the activated oxygen may be released without substrate oxidation through the "leaky" shortcuts in the catalytic cycle (see (1) for a review). This leakage, which results in a futile cycling and generation of reactive oxygen species, such as the superoxide anion-radical and hydrogen peroxide, is closely related to the degree of active site hydration. The mechanisms of conformational gating of the heme pocket that minimize futile cycling through regulation of its solvent accessibility, prevention of aberrant proton delivery and efficient expulsion of the water molecule formed in catalysis represents one of the most important challenges for the current mechanistic enzymology of cytochromes P450. In our search for new approaches to study the conformational dynamics during the...