title: Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance.
abstract: 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 red), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt c ox) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt c red is quantitatively electro-generated from Cyt c ox by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt c red/Cyt c ox can be evaluated accurately. In this study, the values of association/dissociation rate constants (k a, k d) for both COX/Cyt c red and COX/Cyt c ox were obtained. The dissociation constants, K D, were finally calculated as 3.33 × 10(-8) mol · L(-1) for COX/Cyt c red and 4.25 × 10(-5) mol · L(-1) for COX/Cyt c ox, 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. Graphical Abstract Schematic representation of the experimental designs using EC-SPR system. (a) the Au-Cys-COX SPR chip with SAM layers. (b) redox-modulated Cyt c and its binding onto pre-immobilized COX.
authors.affiliation: College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, China.
authors.forename: Yuting
authors.initials: Y
authors.lastname: Hou
authors.affiliation: College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, China.
authors.forename: Jianhong
authors.initials: J
authors.lastname: An
authors.affiliation: College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Deng
authors.affiliation: College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, China. chenshumail@gmail.com.
authors.forename: Shu
authors.initials: S
authors.lastname: Chen
authors.affiliation: College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, China. xiangj@csu.edu.cn.
authors.forename: Juan
authors.initials: J
authors.lastname: Xiang
mesh_terms: D045304:Cytochromes c / Q000032:analysis / Q000737:chemistry*; D004563:Electrochemistry / Q000379:methods*; D003576:Electron Transport Complex IV / Q000032:analysis / Q000737:chemistry*; D004789:Enzyme Activation; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011485:Protein Binding; D025941:Protein Interaction Mapping; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D045304:Cytochromes c; D003576:Electron Transport Complex IV
keywords: Association/dissociation rate constant; Cytochrome c (Cyt c); Cytochrome c oxidase (COX); Electrochemical surface plasmon resonance (EC-SPR); Mimic interface; Redox-dependent interaction