Abstract: | Transitions among various heme coordination/spin states, heme environments and protein conformations of human cytochrome P450 2C8 were investigated under different denaturing conditions by means of electronic absorption and circular dichroism spectroscopies. It is the first report of it's kind. Our results indicated that the thermal and acid‐induced denaturation could convert P450 2C8 to various P420 forms. In the thermal unfolding process, the ferric P420 thermal form emerged with weakened Fe‐S (thiolate) bond. An absorption band at ca. 425 nm of the ferrous P420 2C8 thermal form was observed, suggesting that the axial Cys435 was protonated or displaced by other ligand. Moreover, the new coordination bond was stabilized when the temperature was cooled down. When binding with CO, the ferrous P420 2C8 thermal form had the protonated thiol of Cys435 as the axial ligand. X‐ray structure of P450 2C8 suggested that the specific structure of the β‐bulge where the axial cysteine ligand located might be the reason of the formation of these P420 2C8 thermal forms. In the acid‐induced unfolding studies, we found that at pH 3.0 the heme could be irreversibly released from the heme pocket of ferric and ferrous P450 2C8. Interestingly, the released heme could form a new coordination bond with an unidentified ligand at the surface of partially unfolded protein when binding with CO at reduced state. |