The active site of the thermophilic CYP119 from Sulfolobus solfataricus

CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450 , is stable at up to 85 degrees C. UV-visible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin s tate at higher temperatures. Styrene only causes a small spin state shift, but T-1 NMR studies confirm that styrene is bound in the active site. CYP11 9 catalyzes the H2O2-dependent epoxidation of styrene, cis-beta-methylstyre ne, and cis-stilbene with retention of stereochemistry. This catalytic acti vity is stable to preincubation at 80 degrees C for 90 min. Site-specific m utagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state. Topological analysis by reaction with aryld iazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away. CYP119 is very slowl y reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants. Protein melting curves i ndicate that the thermal stability of CYP119 does not depend on the iron sp in state or the active site architecture defined by the threonine residues. Independence of thermal stability from active site structural factors shou ld facilitate the engineering of novel thermostable catalysts.