Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance
The interactions between the redox couple of cytochrome c (Cyt c) and cytochrome c oxidase (COX) were investigated at a mimic redox-modulated interface by using an electrochemical surface plasmon resonance (EC-SPR) system. Although early studies of the binding between COX and Cyt c have been conducted using several techniques in homogeneous solutions, a problem still inherent is that ferro-cytochrome c (Cyt cred), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt cox) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt cred is quantitatively electro-generated from Cyt cox by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt cred/Cyt cox can be evaluated accurately. In this study, the values of association/dissociation rate constants (ka, kd) for both COX/Cyt cred and COX/Cyt cox were obtained. The dissociation constants, KD, were finally calculated as 3.33 × 10–8 mol · L–1 for COX/Cyt cred and 4.25 × 10–5 mol · L–1 for COX/Cyt cox, respectively. In-situ EC-SPR is promising for better mimicking the in vivo condition that COX is embedded in the inner mitochondrial membrane and Cyt c acts as an electron shuttle in the mobile phase. It is an effective method for the investigation of redox-dependent biomolecular interactions.