Amperometric Response from the Glycolytic versus the Pentose Phosphate Pathway in Saccharomyces cerevisiae Cells Christer F. Spe ´ gel, † Arto R. Heiskanen, † Natalie Kostesha, ‡ Ted H. Johanson, ‡ Marie-F. Gorwa-Grauslund, ‡ Milena Koudelka-Hep, § Jenny Emne ´ us, † and Tautgirdas Ruzgas* ,⊥ Department of Analytical Chemistry, Department of Applied Microbiology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, Institute of Microtechnology, Universite ´ de Neucha ˆ tel, Rue Jaquet Droz 1, 2007 Neucha ˆ tel, Switzerland, and Faculty of Health and Society, Malmo ¨ University, 20506 Malmo ¨ , Sweden The two main metabolic pathways involved in sugar metabolism, i.e., the pentose phosphate pathway (PPP) and the glycolytic pathway (GP), were amperometrically monitored using a double-mediator system composed of menadione and ferricyanide. With the use of the Saccha- romyces cerevisiae deletion mutant, EBY44, lacking the gene encoding for the branch point enzyme phosphoglu- cose isomerize, selective amperometric monitoring of the PPP, mainly producing NADPH, and the GP, mainly producing NADH, could be achieved. It was found that the bioelectrocatalytic current was primarily originating from NADPH. This conclusion was supported by metabo- lite flux analysis, confirming that, in the presence of menadione, the cells increase the rate of NADPH-produc- ing reactions although these processes might be detri- mental to cell survival. The higher rate of in vivo NADPH- dependent menadione reduction can be ascribed to the fact that the intracellular NADPH/NADP + ratio is much higher than NADH/NAD + as well as that the former ratio is more tightly controlled. This tight control over the cofactor ratios is lost upon cell disintegration as observed from spectrophotometric assays using crude cell extract, and amperometric investigations of permeabilized cells indicate a higher rate of NADH- than NADPH-dependent menadione reduction. These in vitro experiments show a higher activity of NADH-dependent than NADPH-depend- ent menadione-reducing dehydrogenases in S. cerevisiae cells. Bioelectrochemistry, as a research discipline, is strongly acknowledged by its contribution to the development of ampero- metric enzyme-based biosensors, e.g., glucose biosensors. 1,2 A number of new applications are expected from fundamental studies of electron transfer (ET) reactions of redox enzymes at conducting materials of macro 3 or nanoscopic 4,5 dimensions as well as in enzyme redox hydrogel structures. 6 Electrochemical investigation of redox processes in living cells are also carried out. Direct ET between intact living cells and electrodes has been observed and is under growing fundamental interest. 7-10 The majority of the electrochemical measurements on living cells, however, address biochemical redox processes by using redox mediators, which shuttle electrons between the electrode and intracellular redox reactions. 11-18 Following the progress in cell biology, it is of great interest to understand and demonstrate how electrochemical techniques can be exploited, e.g., to monitor a particular intra- cellular redox process or defined metabolic and signaling pathway or to assay a specific enzyme under in vivo conditions. Exciting examples in this direction are measurements of enzyme activity in living cells, e.g., hydrogenase activity, 15 alcohol dehydrogenase activity, 14 and study of detoxification of menadione by cells. 16,19 * Corresponding author. Phone: +46-40-6657431. Fax: +46-40-6658100. 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J.; Owhadian, O.; Monks, T. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17582-17587. Anal. Chem. 2007, 79, 8919-8926 10.1021/ac0710679 CCC: $37.00 © 2007 American Chemical Society Analytical Chemistry, Vol. 79, No. 23, December 1, 2007 8919 Published on Web 11/01/2007