Knowledge of the relationship between the activity and structural features of enzymes is critical for designing conjugation materials. Nonetheless, few studies have addressed the decrease in enzyme activity after modifications of the corresponding enzymes. In cytochrome P450cam, the structural changes around the heme active center can be easily evaluated by spectrophotometric analysis. Thus, with the use of P450cam, we evaluated the relationship between enzyme activity and structural changes after its modification. Our study revealed the existence of a new mode for P450 activity downregulation, which was distinct from a well-known inactivation mode for the conversion of P450 to the inactive P420 form. In this new mode, the activity decreased due to a reduction in binding or accessibility of a substrate to the heme pocket of P450cam, while the enzymatic tertiary structure was retained after conjugations.