Evolving a New Electron Transfer Pathway for Nitrogen Fixation Uncovers an Electron Bifurcating-Like Enzyme Involved in Anaerobic Aromatic Compound Degradation

Abstract

Nitrogenase is the key enzyme involved in nitrogen fixation and uses low potential electrons delivered by ferredoxin (Fd) or flavodoxin (Fld) to reduce dinitrogen gas (N₂) to produce ammonia, generating hydrogen gas (H₂) as an obligate product of this activity. Although the phototrophic alphaproteobacterium Rhodopseudomonas palustris encodes multiple proteins that can reduce Fd, the FixABCX complex is the only one shown to support nitrogen fixation, and R. palustris Fix^- mutants grow poorly under nitrogen-fixing conditions. To investigate how native electron transfer chains (ETCs) can be redirected toward nitrogen fixation, we leveraged the strong selective pressure of nitrogen limitation to isolate a suppressor of an R. palustris ΔfixC strain that grows under nitrogen-fixing conditions. We found two mutations were required to restore growth under nitrogen-fixing conditions in the absence of functional FixABCX. One mutation was in the gene encoding the primary Fd involved in nitrogen fixation, fer1, and the other mutation was in aadN, which encodes a homolog of NAD⁺-dependent Fd:NADPH oxidoreductase (Nfn). We present evidence that AadN plays a role in electron transfer to benzoyl coenzyme A reductase, the key enzyme involved in anaerobic aromatic compound degradation. Our data support a model where the ETC for anaerobic aromatic compound degradation was repurposed to support nitrogen fixation in the ΔfixC suppressor strain. IMPORTANCE There is increasing evidence that protein electron carriers like Fd evolved to form specific partnerships with select electron donors and acceptors to keep native electron transfer pathways insulated from one another. This makes it challenging to integrate a Fd-dependent pathway such as biological nitrogen fixation into non-nitrogen-fixing organisms and provide the high-energy reducing power needed to fix nitrogen. Here, we show that amino acid substitutions in an electron donor for anaerobic aromatic compound degradation and an Fd involved in nitrogen fixation enabled electron transfer to nitrogenase. This study provides a model system to understand electron transfer chain specificity and how new electron transfer pathways can be evolved for biotechnologically valuable pathways like nitrogen fixation.

Keywords: NAD+-dependent ferredoxin:NADPH oxidoreductase; Rhodopseudomonas palustris; ferredoxin; nitrogenase.

FIG 1

Current model of electron transfer to nitrogenase (N₂ase) and benzoyl-CoA reductase (BCR) in R. palustris. Electron transfer to BCR is incompatible with nitrogen fixation in the wild type, but the C38W substitution in AadN and T11I substitution in Fer1 enable the electron transfer pathway for benzoate degradation to support nitrogen fixation in ΔfixC*. The hypothesized activity of AadN shown in dotted lines is inferred based on its similarity to NAD⁺-dependent ferredoxin:NADPH oxidoreductase from Pyrococcus furiosus.

FIG 2

Growth of ΔfixC* in nitrogen-fixing conditions does not require fixA but does require fer1^T11I. (A) Growth of wild-type R. palustris CGA753 (WT), R. palustris with a deletion in fixC (ΔfixC), R. palustris suppressor of ΔfixC (ΔfixC*), and R. palustris suppressor of ΔfixC with a deletion in fixA (ΔfixC* ΔfixA) in minimal medium lacking ammonium sulfate (nitrogen-fixing) with 20 mM acetate. (B) Growth of wild-type R. palustris CGA753 (WT), R. palustris with a deletion in fixC (ΔfixC), R. palustris suppressor of ΔfixC (ΔfixC*), R. palustris suppressor of ΔfixC with a deletion in fer1 (ΔfixC* Δfer1), R. palustris suppressor of ΔfixC with a deletion in fldA (ΔfixC* ΔfldA), and R. palustris suppressor of ΔfixC with a deletion in fer1 and fldA (ΔfixC* Δfer1 ΔfldA) in minimal medium lacking ammonium sulfate (nitrogen-fixing) with 20 mM acetate. For both panels A and B, the data are averages of two biological replicates, and error bars represent one standard deviation from the mean.

FIG 3

ΔfixC* alleles of fer1 and aadN enable growth of ΔfixC in nitrogen fixing conditions. Growth of R. palustris suppressor of ΔfixC (ΔfixC*), R. palustris with a deletion in fixC (ΔfixC), R. palustris ΔfixC encoding a fer1^T11I allele (ΔfixC fer1^T11I), R. palustris ΔfixC encoding an aadN^C38W allele (ΔfixC aadN^C38W), and R. palustris ΔfixC encoding the fer1^T11I and aadN^C38W alleles (ΔfixC fer1^T11I aadN^C38W) in minimal medium lacking ammonium sulfate (nitrogen-fixing) supplemented with 20 mM acetate. Data shown are the average of three biological replicates, error bars represent one standard deviation from the mean.

FIG 4

AadN, an Nfn homolog, is required for anaerobic aromatic compound degradation. (A) AadN is homologous to NfnI from P. furiosus (PfNfnI) and a pattern B Nfn from Clostridium autoethanogenum (CaNfn). The percent amino acid identity of the large and small subunits of PfNfnI and CaNfn to AadN are shown over the small (green) and large (orange) regions. (B) PfNfnI ligates two [4Fe-4S] clusters, one [2Fe-2S] cluster, and two flavin adenine dinucleotide (FAD) cofactors per PfNfnI heterodimer. The amino acid sequence of the binding domains for each of these cofactors is conserved in AadN, but it remains unclear if AadN interacts with the same redox pools as PfNfnI. (C) Map of genomic region around aadN in R. palustris CGA009 shows that aadN is near genes involved in aromatic compound degradation (hba genes, cyan). (D) Growth phenotypes of R. palustris CGA753 (WT), R. palustris with a deletion in aadN (ΔaadN), and R. palustris with a deletion in fixC and encoding the aadN^C38W allele (ΔfixC aadN^C38W) in minimal medium supplemented with 10 mM HCO₃ and either 5.7 mM benzoate (BA), 5.7 mM 4-hydroxybenzoate (4-HB), or 5.7 mM cyclohexane carboxylate (CHC) as carbon sources. Cultures shown are representative of three independent trials. (E) Growth phenotypes of R. palustris CGA753 (WT), R. palustris with a deletion in fixC (ΔfixC), R. palustris with a deletion in aadN (ΔaadN) and R. palustris with a deletion in both fixC and aadN (ΔΔ) in minimal medium lacking ammonium sulfate (nitrogen-fixing) supplemented with 20 mM acetate (AC) or 5.7 mM benzoate (BA). Cultures shown are representative of three independent trials.