Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological function of neutral red in membranedriven fumarate reduction and energy conservation

Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes pu-rified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell mem-branes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activ-ities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hy-drogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glu-cose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20 % while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by puri-fied membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinoline N-oxide to whole cells or purified membranes inhibited succinate production from H2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes t