Volume 17, number 2 FEBS LEITERS October 1971 zyxwvutsr POLAROGRAPHIC ACTIVITY AND ELECTROLYTIC REDUCTION OF FERREDOXIN P.D.J. WEITZMAN, I.R. KENNEDY and R.A. CALDWELL Department of Biochemistry, University of Leicester, Leicester, LEI 7RH, England and Department of Agricultural Chemistry, University of Sydney, Sydney, Australin Received 20 July 197 1 1. Introduction The structure and function of ferredoxin have been the subject of intensive investigation. Its physiologi- cal participation in electron transfer, coupled with our interest in polarography as a technique for stu- dying redox systems prompted an examination for ferredoxin polarographic activity. In this communi- cation we report that ferredoxin does give a charac- teristic polarographic signal, that this is associated with the biological function of the protein and that electrolytic reduction may offer an advantageous means of preparing reduced ferredoxin. To our know- ledge no studies of the analytical or preparative elec- troreduction of ferredoxin have previously been re- ported. 2. Experimental Ferredoxin was purified from Clostridium pasteu- rianum by the method described, and to the purity indicated, by Mortenson [l] . Polarographic measure- ments were made with a Radiometer PO4 recording polarograph using a reaction vessel and procedure as previously described [2]. The dropping mercury elec- trode was employed, together with the saturated calomel reference electrode (S.C.E.). The polarograph was used at high sensitivity, in the range 0.1-0.3 yA full-scale deflection, at which sensitivity polarograms of buffer solutions themselves have a high slope. To overcome this, condenser-current compensation was applied, usually to the extent of 0.2 PA/V. Electro- lytic reduction was carried out by the method de- scribed by Cecil and Weitzman [3] or a modifica- tion thereof as follows. It was desirable to carry out polarographic measurements and electroreduction in the same apparatus, thereby obviating the need to transfer material from one vessel to another, with consequent risk of some oxidation of the labile re- duced ferredoxin. This was achieved by using a po- larographic cell as previously described [2] modi- fied to contain a platinum wire fused through the bottom and making contact with a small pool of mercury in the cell. A small magnetic flea floated on the surface of the mercury and, during electro- reduction, was rotated to stir the mercury surface. By this means it was possible to make analytical polarographic measurements by applying potentials between the dropping mercury electrode and refer- ence calomel electrode, or to carry out preparative electroreduction by applying a potential (from an external battery or the polarograph itself) between the stirred mercury pool cathode and the calomel anode. The apparatus could readily be switched from one mode of operation to the other. 3. Results and discussion C. pasteurianum ferredoxin was found to give a well-defined polarographic reduction wave, as shown in fig. 1. The half-wave potential at pH 7 was -570 mV (versus the S.C.E.), equivalent to -325 mV North-Holland Publishing Company - Amsterdam 241