Surface charge-controlled electron transfer and catalytic behavior of immobilized cytochrome P450 BM3 inside dendritic mesoporous silica nanoparticles

Understanding the influencing factors on the reaction kinetics of P450 BM3 within confined spaces is essential for developing efficient P450 BM3 bioreactors. Herein, two dendritic mesoporous silica nanoparticles (OH-DMSNs and NH₂-DMSNs) with similar pore size but opposite surface charge have been prepared and served as the vehicle to immobilize P450 BM3. With the help of the film-forming material of chitosan, P450 BM3/OH-DMSN and P450 BM3/NH₂-DMSN composites were immobilized on GC electrode and characterized with electrochemical measurements. Compared with P450 BM3/OH-DMSNs/GCE, P450 BM3/NH₂-DMSNs/GCE showed higher electron transfer efficiency with higher current charge and lower k_s value. Besides, the generated catalytic current towards testosterone on P450 BM3/NH₂-DMSNs/GCE was 1.81 times larger than P450 BM3/OH-DMSNs/GCE. Furthermore, P450 BM3 inside NH₂-DMSNs displayed higher affinity towards testosterone with the lower K_m^app value of 244.82 μM. These results are attributed to the positively charged internal walls of NH₂-DMSNs so that P450 BM3 adapts to an orientation favorable for electron exchange with electrodes and substrate binding with the active sites. The present study provides fundamentals for regulating the surface charge to optimize redox process and catalytic behavior in CYP bioreactors through electrostatic interactions.