There is significant biotechnological interest in the engineering of nitrogenase into plants. To succeed, metalloclusters need to be formed and loaded into the active sites of various components of nitrogenase pathways. A traditional model of nitrogenase biogenesis places NifU as the central “server” of 4Fe4S clusters that are transferred to downstream “clients”, including apo-NifH and apo-AnfH. Given the complex genetics of nitrogenase assemblies perhaps the most conclusive evidence of this model is in vitro biochemical assays using purified enzymes where the 4Fe4S clusters on NifU are rapidly transferred to apo-NifH forming active NifH.
The obligate requirement for NifU for loading apoH has been challenged in both yeast and bacterial models. In the mitochondria of yeast both Azotobacter NifH and AnfH were found to be active ‘as isolated’ without co-expression of NifSU. These results in yeast are mirrored also in E. coli where transgenic expression of both Klebsiella NifH and Azotobacter NifH can be isolated as active and loaded with 4Fe4S clusters also in the absence of NifSU. The results from both E. coli and yeast indicate that NifSU is not the exclusive pathway for loading apoH and that non-NifSU endogenous pathways are capable of loading these transgenic enzymes with 4Fe4S clusters.
Here we present our results regarding the functionality of NifH and AnfH expressed in the mitochondria of plant leaves. We demonstrate that a soluble version of Klebsiella NifH was only partially active ‘as isolated’ and this activity was reduced even further when co-expressed with NifSU. Similarly, a soluble version of Azotobacter AnfH was not active as isolated with co-expression with NifSU. Both versions of leaf-isolated NifH and AnfH were capable of accepting 4Fe4S clusters when donated in vitro from NifU. These results indicate that the transgenic proteins were correctly folded in the plant mitochondria, but were essentially lacking clusters.
We will briefly outline the approaches we have taken to express soluble nitrogenase proteins in plants combined with biochemical characterisation of the isolated proteins, and discuss our results in plants relative to bacteria- and yeast-based assays in other labs.