On the energy-coupling sites inParacoccus denitrificans

Cell-free preparations fromParacoccus denitrificans grown aerobically under autotrophic and heterotrophic conditions catalyzed succinate- and ascorbate-linked ATP-dependent NAD⁺ reduction. The succinate-linked NAD⁺ reduction was sensitive to rotenone and indicated the participation of coupling site 1. The ascorbate-linked, energy-driven, reversed electron flow was sensitive to antimycin A or 2-n-heptyl-4-hydroxyquinoline-N-oxide and rotenone, thus indicating the mediation of coupling sites 2 and 1. The aerobic oxidation of three ascorbate equivalents was required to reduce one NAD⁺ equivalent in the autotrophic system, while no NAD⁺ reduction coupled to ascorbate oxidation occurred in the heterotrophic system unlessN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was present. In the later case, the oxidation of 24 equivalents of reduced TMPD was required to support the reduction of each equivalent of NAD⁺ These results indicated that, while there was an efficient energy-coupling at site 3 in the autotrophic system, the heterotrophic system appeared to posses a very weak or inefficient terminal energy-coupling site.