D-Fructose dehydrogenase (FDH) is a heterotrimeric-membrane-bound enzyme capable of direct electron transfer (DET)-type bioelectrocatalysis. Subunit II contains three heme C moieties and is presumed to play a key role in the DET reaction because the subunit I/III subcomplex (without subunit II) lacks DET-type activity. We constructed an expression system for subunit II. A non-turnover signal from subunit II was not observed on voltammograms, and a subunit II-adsorbed electrode did not exhibit DET-type activity in the presence of the subunit I/III subcomplex and fructose. Gel filtration column chromatography indicated that subunit II formed multimeric complexes with four or more subunits. The aggregation of subunit II seemed to interfere with its direct communication with the electrodes. In contrast, when subunit II was mixed with the subunit I/III complex in a solution, they formed a 1:1 full complex, and the complex recovered DET-type bioelectrocatalytic activity. These results strongly suggest that the heme C in subunit II is the electron transfer site for the DET-type bioelectrocatalytic activity of FDH. (C) 2016 Elsevier Ltd. All rights reserved.