Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

Abstract

A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.nitrogen fixation | electron transport | plant organelles | nitrogenase engineering

Physiological responses of the freshwater N2‐fixing cyanobacterium Raphidiopsis raciborskii to Fe and N availabilities

“…For example, N 2 -fixation provides an essential N source to support the relatively low growth and cell homeostasis of R. raciborskii under prolonged N-limited conditions (Willis et al, 2016a). N 2 -fixation by most diazotrophs is mediated by nitrogenase with a cofactor containing iron (Fe) and other transition metals (Mo and V) (Yang et al, 2017). N 2 -fixation by most diazotrophs is mediated by nitrogenase with a cofactor containing iron (Fe) and other transition metals (Mo and V) (Yang et al, 2017).…”

“…These facultative characteristics also provide competitive growth advantages for R. raciborskii under conditions where combined N concentrations fluctuate (Moisander et al, 2012;Burford et al, 2016). This metalloenzyme is composed of two component proteins, namely dinitrogenase (an Fe-X protein where X represents Fe, Mo or V) and dinitrogenase reductase (an Fe protein) (Yang et al, 2017). This metalloenzyme is composed of two component proteins, namely dinitrogenase (an Fe-X protein where X represents Fe, Mo or V) and dinitrogenase reductase (an Fe protein) (Yang et al, 2017).…”

“…N 2 -fixation by most diazotrophs is mediated by nitrogenase with a cofactor containing iron (Fe) and other transition metals (Mo and V) (Yang et al, 2017). This metalloenzyme is composed of two component proteins, namely dinitrogenase (an Fe-X protein where X represents Fe, Mo or V) and dinitrogenase reductase (an Fe protein) (Yang et al, 2017). Nitrogenase synthesis requires a large amount of Fe [25-39 atoms of Fe per nitrogenase molecule (Postgate, 1998) with 236 μmol of Fe bound to nitrogenase per mol of cellular carbon (Whittaker et al, 2011)].…”

Keeping the nitrogen-fixation dream alive

“…It is therefore anticipated that an active dinitrogenase can also be produced in such experimental systems if the appropriate assembly requirements are satisfied. It also appears that the much anticipated "oxygen problem" might be circumvented by mitochondrial targeting or, perhaps, plastid-targeting strategies combined with controlled conditions for expression, as suggested by Yang et al (7). Second, dinitrogenase reductase contains an ironsulfur cofactor that is essential for its activity (10).…”

“…NifJ couples the oxidation of pyruvate to the reduction of NifF, which, in turn, serves as a direct electron donor to dinitrogenase reductase. Yang et al substituted NifF by various electron carriers of plastid origin, known as ferredoxins, and obtained functional hybrid electron-transfer modules (7). In this case the functional unit required that NifJ remain intact; thus, a hybrid module consisting of a bacterial protein and a plastid protein is effective in coupling cellular metabolism to nitrogen fixation.…”