Abstract
The vnf-encoded apodinitrogenase (apodinitrogenase 2) has been purified from Azotobacter vinelandii strain CA117.30 (DeltanifKDB), and is an alpha2beta2delta2 hexamer. Apodinitrogenase 2 can be activated in vitro by the addition of the iron-vanadium cofactor (FeV-co) to form holodinitrogenase 2, which functions in C2H2, H+, and N2 reduction. Under certain conditions, the alpha2beta2delta2 hexamer dissociates to yield the free delta subunit (the VNFG protein) and a form of apodinitrogenase 2 that exhibits no C2H2, H+, or N2 reduction activities in the in vitro FeV-co activation assay; however, these activities can be restored upon addition of VNFG to the FeV-co activation assay system. No other vnf-, nif-, or non-nif-encoded proteins were able to replace the function of VNFG in the in vitro processing of alpha2beta2 apodinitrogenase 2 (in the presence of FeV-co) to a form capable of substrate reduction. Apodinitrogenase 2 is also activable in vitro by the iron-molybdenum cofactor to form a hybrid enzyme with unique properties, most notably the inability to reduce N2 and insensitivity to CO inhibition of C2H2 reduction.
Highlights
The biological conversion of atmospheric nitrogen to ammonia can occur via three distinct nitrogenase enzymes in Azotobacter vinelandii: the conventional molybdenum-containing enzyme, a vanadium-containing enzyme, and a third enzyme that is believed to contain only iron
The role of the ␦ subunit in the functioning of dinitrogenase 2 was recently addressed by Waugh et al, who found that an A. vinelandii strain lacking vnfG was unable to grow diazotrophically, suggesting that the ␦ subunits might be required for N2 reduction [11]
The requirement for specific polypeptide-cofactor interactions was observed with dinitrogenase 1 containing the iron-vanadium cofactor (FeV-co);1 the hybrid enzyme was unable to reduce N2, and C2H2 was reduced to both C2H4 and C2H6, the latter being a property of nitrogenase 2 [19]
Summary
FeV-co, iron-vanadium cofactor; FeMoco, iron-molybdenum cofactor; FeFe-co, iron-iron cofactor; HPLC, high pressure liquid chromatography; DTH, sodium dithionite; PAGE, polyacrylamide gel electrophoresis. Vnf-encoded Apodinitrogenase from A. vinelandii composition and is activable in vitro by FeV-co. The ␦ subunit (VNFG) dissociates from the ␣22␦2 hexamer to yield free VNFG and a form of apodinitrogenase 2 that is inactive in the in vitro FeV-co-activation assay. Apodinitrogenase 2 activity can be restored upon addition of VNFG to the FeV-co-activation assay system. In addition to being activable by FeV-co, apodinitrogenase 2 is activable in vitro by FeMo-co. The activation of apodinitrogenase 2 by FeFe-co (the putative iron-only cofactor of dinitrogenase 3) was attempted; the substrate reduction characteristics of the resulting hybrid enzymes were investigated
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