The enigmatic enzyme 'amidoxime reducing component' of Lotus japonicus. Characterization, expression, activity in plant tissues, and proposed role as a nitric oxide-forming nitrite reductase.

Abstract

Human mitochondria contain a molybdoprotein capable of reducing amidoximes using cytochrome b5/cytochrome b5 reductase (Cb/CbR). This 'amidoxime reducing component' (ARC) also reduces nitrite to nitric oxide (NO). In the plant kingdom, distinct functions have been suggested for ARCs. Thus, the single ARC of Chlamydomonas reinhardtii (crARC) reduces nitrite to NO by taking electrons from nitrate reductase (NR). Therefore, it was proposed that a dual NR/crARC system can generate NO under physiological conditions and the crARC was renamed to 'NO-forming nitrite reductase' (NOFNiR). In contrast to this, the two ARC enzymes from Arabidopsis thaliana were not found to produce NO in vitro at physiological nitrite concentrations, suggesting a different, as yet unknown, function in vascular plants. Here, we have investigated the two ARCs of Lotus japonicus (LjARCs) to shed light on this controversy and to examine, for the first time, the distribution of ARCs in plant tissues. The LjARCs are localized in the cytosol and their activities and catalytic efficiencies, which are much higher than those of A. thaliana, are consistent with a role as NOFNiR. LjARCs are prone to S-nitrosylation in vitro by S-nitrosoglutathione and this post-translational modification drastically inhibits their activities. The enzymes are mainly expressed in flowers, seeds and pods, but are absent in nodules. LjARCs are active with NR and Cb/CbR as electron-transferring systems. However, the LjNR mRNA levels in seeds and pods are negligible, whereas our proteomic analyses show that pods contain the two ARCs, Cb and CbR. We conclude that LjARCs may play a role as NOFNiR by receiving electrons from the Cb/CbR system but do not act in combination with NR.