Role of the C-terminal extension stacked on the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group in ferredoxin-NADP+ oxidoreductase from Bacillus subtilis

Ferredoxin-NADP+ oxidoreductase [EC 1.18.1.2] from Bacillus subtilis (BsFNR) is homologous to the bacterial NADPH-thioredoxin reductase, but possesses a unique C-terminal extension that covers the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide (FAD) prosthetic group. In this report, we utilize BsFNR mutants depleted of their C-terminal residues to examine the importance of the C-terminal extension in reactions with NADPH and ferredoxin (Fd) from B. subtilis by spectroscopic and steady-state reaction analyses. The depletions of residues Y313 to K332 (whole C-terminal extension region) and S325 to K332 (His324 intact) resulted in significant increases in the catalytic efficiency with NADPH in diaphorase assay with ferricyanide, whereas Km values for ferricyanide were increased. In the cytochrome c reduction assay in the presence of B. subtilis ferredoxin, the S325–K332 depleted mutant displayed a significant decrease in the turnover rate with an Fd concentration range of 1–10 μM. The Y313–K332 depleted mutant demonstrated an increase in the rate of the direct reduction of horse heart cytochrome c in the absence of Fd. These data indicated that depletion of the C-terminal extension plays an important role in the reaction of BsFNR with ferredoxin.