Utilization of electrically reduced neutral red by Actinobacillus succinogenes: Physiological function of neutral red in membrane-driven fumarate reduction and energy conservation

Neutral red (NR) functioned as an electronophore or electron channel enabli ng either cells or membranes purified from Actinobacillus succinogenes to d rive electron transfer and proton translocation by coupling fumarate reduct ion to succinate production. Electrically reduced NR, unlike methyl or benz yl viologen, bound to cell mem branes, was not toxic, and chemically reduce d NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and dia phorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the hig hest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dep endent on either electrically reduced NR or H-2 as the electron donor and o n the fumarate concentration, During the growth of Actinobacillus on glucos e plus electrically reduced NR in an electrochemical bioreactor system vers us on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate pr oduction by about 50%. The rate of fumarate reduction to succinate by purif ied membranes was twofold higher with electrically reduced NR than with hyd rogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinolin e N-oxide to whole cells or purified membranes inhibited succinate producti on from H-2 plus fumarate but not from electrically reduced NR plus fumarat e. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power.