RESPIRATORY ELECTRON-TRANSFER ACTIVITY IN AN ASOLECTIN-ISOOCTANE REVERSE MICELLAR SYSTEM

Bovine heart submitochondrial particles (SMP) were solubilized in an a solectin isooctane reverse micellar system and the functionality of th e respiratory chain was tested by spectroscopic and amperometric techn iques. Electron transfer rate supported by NADH was very slow as evide nced by the low cytochrome reduction levels attained over long incubat ion periods. In the presence of KCN, NADH caused 34% and 12.5% reducti on of the cytochromes aa3 and c, respectively, and negligible reductio n of cytochrome b. Supplementation of the system with menadione rose t he NADH-dependent reduction of all the cytochromes to levels that were close to the total content. However, no measurable O2 uptake activity took place in the presence of NADH plus menadione, or with ascorbate (or NADH) plus TMPD reducing systems. Therefore, it is suggested that in the organic medium, electron transfer from NADH to O2 is arrested a t the terminal oxidase step. Cytochrome oxidase reduced by ascorbate ( or NADH) plus TMPD seems to be trapped in its half reduced state (ie, a2+ a3(3+)). Although it is poorly reactive with O2, it can transfer e lectrons back to cytochrome c and TMPD. The electron transfer block to O2 was overcome when PMS was used instead of TMPD. This seems to be d ue to the recognized capacity of PMSH2 to carry out simultaneous reduc tion of both a CuA and a3 CuB redox centers of cytochrome oxidase. The cytochrome oxidase reaction in the organic solvent was highly sensiti ve to KCN (K(i) 1.9-mu-M) and showed bell-shaped kinetics towards the PMS concentration and a sigmoidal response to water concentration, rea ching its maximal turnover number (18 s-1) at 4 mM PMS and 1.1% (v/v) water. This value was about 92% and 54% of that attained with the nati ve and reconstituted enzyme, respectively, in totally aqueous assays.