Kinetic characterization of succinate‐dependent plasma membrane‐bound nitrate reductase in tobacco roots 1

Abstract

Plasma membrane‐bound nitrate reductase (PM‐NR) of tobacco (Nicotiana tabacum L. cv. Samsun) roots reduces nitrate with NADH and/or succinate as electron donor. The present paper reports on significant differences of the succinate‐dependent activity (succ‐PM‐NR) to known NADH‐dependent soluble and plasma membrane‐bound NR. The experiments were performed with plasma membrane vesicles containing the hydrophobic PM‐NR. In a temperature course, succ‐PM‐NR activity attained the highest rates of total activity (succinate‐nitrate) at 50°C, NADH‐dependent PM‐NR (NADH‐PM‐NR) only at 30°C. The temperature responses of partial reactions with domains of NR diverged, but indicated that the heme domain was the most sensitive to high temperatures and could limit succ‐PM‐NR. In contrast to NADH, succinate did not supply electrons to ferricyanide reduction. The pH optima of the overall reaction with succinate were 5.6 and 8.0 at 30°C, pH 7.0 at 50°C, with a much higher rate in the latter case. The affinities of succ‐PM‐NR for both substrates (succinate and nitrate) were highest at pH 5.6 and higher at 50°C than at 30°C. Malonate showed competitive inhibition with succinate, but not with NADH as substrate. We assume that structural differences in the flavin domain of at least one of the subunits of root PM‐NR may be responsible for the succinate‐dependent in addition to the NADH‐dependent activity, but a more detailed analysis is necessary.