The structure-function relationship of cytochrome c (Cyt-c) interacted with the lipid bilayer membranes was studied by various spectroscopic methods, the reaction-products analysis, and its kinetics. Ultrafiltration binding assay, UV-visible, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies showed that Cyt-c was tightly bound to the lipid bilayer membranes bearing a phosphate head group. The anisotropic and nonnatural complexation with the phosphate-lipid membranes caused a spin-state change of the heme in the active center of Cyt-c. Depending on the membrane fluidity, two classes of the structurally altered Cyt-c were prepared and they showed the greatly enhanced N-demethylase activity. Products analysis by HPLC demonstrated that the lipid membrane bound Cyt-c performs a clean enzymatic reaction similar to native hemoenzymes. Kinetics studies established that there are two different activation manners via the phosphate lipid bilayer membranes: namely, simple enhancement of the affinity for H2O2, or the increased catalytic efficiency (k(cat)) in addition to the enhanced affinity for H2O2. The membrane fluidity again significantly affected the N-demethylation kinetics. A potential of the lipid membrane assembly to functionalize native proteins and enzymes with noncovalent but specific interactions is also discussed.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry