Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance.

Hou, Yuting; An, Jianhong; Deng, Chunyan; Chen, Shu; Xiang, Juan

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 c), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt c) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt c is quantitatively electro-generated from Cyt c by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt c/Cyt c can be evaluated accurately. In this study, the values of association/dissociation rate constants ( k, k) for both COX/Cyt c and COX/Cyt c were obtained. The dissociation constants, K, were finally calculated as 3.33 × 10 mol · L for COX/Cyt c and 4.25 × 10 mol · L for COX/Cyt c, 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. [Figure not available: see fulltext.]

CYTOCHROME oxidase; SURFACE plasmon resonance; MITOCHONDRIAL membranes; ELECTRONS; OXIDATION-reduction reaction

Analytical & Bioanalytical Chemistry, 2016, Vol 408, Issue 18, p4935

1618-2642

Academic Journal

10.1007/s00216-016-9586-9