Heterologous expression of a fully active Azotobacter vinelandii nitrogenase Fe protein in Escherichia coli

ABSTRACT The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii (AvNifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active AvNifH protein upon co-expression of this protein with AvIscS/U and AvNifM in E. coli. Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed AvNifH protein has a high [Fe4S4] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that AvNifM could serve as a chaperone and assist the maturation of a cluster-replete AvNifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the “clean” genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

cule activation under ambient conditions (1)(2)(3).Recognized mainly for its role as the reductase component of nitrogenase, the Fe protein of the Mo-dependent nitroge nase from the diazotrophic bacterium Azotobacter vinelandii (designated AvNifH) is an ~60 kDa homodimer that features a subunit-bridging [Fe 4 S 4 ] cluster and an MgATP-bind ing site within each subunit (1)(2)(3)(4)(5)(6)(7).In the nitrogenase reaction, AvNifH works in concert with its catalytic partner, MoFe protein (designated AvNifDK), to enable ATP-dependent electron transfer from the [Fe 4 S 4 ] cluster of AvNifH, through the [Fe 8 S 7 ] P-cluster, to the [(R-homocitrate)-MoFe 7 S 9 C] M-cluster of AvNifDK, where the reduction of various substrates, such as N 2 , H + , C 2 H 2 , and CO, occurs under ambient conditions (Fig. 1A) (3,5,(8)(9)(10)(11)(12)(13).In addition to its essential role as an electron donor to its catalytic partner during substrate reduction, AvNifH can act as an independent reductase and catalyze the in vivo and in vitro transformation of CO 2 to CO at its [Fe 4 S 4 ] center in the presence or absence of ATP (Fig. 1B) (14,15).Extending its functions from catalysis to biosynthe sis, AvNifH also plays a key role in the maturation of both P-and M-clusters, the two unique, high-nuclearity metal centers within AvNifDK that are central to the reactivity of nitrogenase.Regarding P-cluster assembly, AvNifH facilitates the coupling of a [Fe 4 S 4 ] cluster pair into a P-cluster at the α/β-subunit interface of AvNifDK (Fig. 1C) (16)(17)(18)(19).In the context of M-cluster assembly, AvNifH assists in inserting Mo and homocitrate (20)(21)(22) into an [Fe 8 S 9 C] precursor on AvNifEN (23,24), a biosynthetic protein with strong sequence and structural resemblance to AvNifDK (25), to generate a mature M-cluster that is subsequently delivered to the cofactor-binding site within AvNifDK (Fig. 1D) (3,26).In both biosynthetic processes, AvNifH likely interacts with its assembly partners in a way that mirrors its interaction with its catalytic partner during substrate turnover, functioning as an ATP-dependent reductase to enable the maturation of the complex P-and M-clusters of AvNifDK.
The ability of the Fe protein to act as a multifunctional reductase hinges on the capability of its [Fe 4 S 4 ] cluster to undergo facile redox changes.In the case of AvNifH, its [Fe 4 S 4 ] cluster is known to adopt three different oxidation states: the oxidized state ([Fe 4 S 4 ] 2+ ), reduced state ([Fe 4 S 4 ] 1+ ), and the super-reduced, all-ferrous state ([Fe 4 S 4 ] 0 ) (1,2,11,27).While ferredoxins or flavodoxins are likely to function as the physiological electron donors for AvNifH under in vivo conditions, chemical reductants are typically used as the artificial electron donors for AvNifH under in vitro conditions (3)(4)(5)11).An example of the chemical reductants is Eu II -DTPA (with E 1/2 = −1.14V at pH 8) (2,28,29), which is employed with or without ATP in the in vitro reduction of CO 2 by AvNifH.In this process, the [Fe 4 S 4 ] cluster of AvNifH exists in the all-ferrous ([Fe 4 S 4 ] 0 ) state, which can undergo a reversible one-or two-electron redox transition of the cluster to the reduced ([Fe 4 S 4 ] 1+ ) or oxidized ([Fe 4 S 4 ] 2+ ) state to enable substrate reduction (14,30).Another commonly used chemical reductant is dithionite (e.g., with E 1/2 = −0.47V at 2 mM dithionite at pH 8), which is used along with MgATP to facilitate the in vitro reduction of N 2 by the complete nitrogenase enzyme.In this case, the [Fe 4 S 4 ] cluster of AvNifH is maintained in the reduced ([Fe 4 S 4 ] 1+ ) state, which can undergo a reversible one-electron redox reaction to the oxidized ([Fe 4 S 4 ] 2+ ) state to enable ATP-dependent electron transfer to the active-site cofactor of AvNifDK for substrate reduction (11).It should be noted that the binding of MgATP induces a conformational change of AvNifH concomitant with a decrease of the midpoint potentials of its associated [Fe 4 S 4 ] cluster by 140 mV (to ca.−430 mV) (4), which facilitates the protein-protein interaction between AvNifH and AvNifDK while promoting the inter-protein transfer of electrons from the former to the latter (1)(2)(3)(4)(5)(6)11).Interestingly, while AvNifH interacts with its catalytic and assembly partners in an analogous, ATP-dependent manner, a recent study using a [Fe 4 Se 4 ] substituted AvNifH as a probe has revealed a difference in the redox requirements of these events, with the catalytic reactions by nitrogenase necessitating a lower reduction potential than the assembly processes of its metalloclusters (29).
The functional versatility of Fe protein makes it an appealing target for heterologous expression in a genetically amenable host like Escherichia coli, which could prove useful for developing nitrogenase-based biotechnological adaptations for the production of valuable chemical commodities in the future.Yet, the heterologous synthesis of a fully functional Fe protein in E. coli has thus far met with mixed results.Previous efforts have led to the heterologous expression and partial purification of the Klebsiella pneumoniae NifH protein from E. coli, as well as the successful purification and characterization of the methanogen NifH protein expressed in the same heterologous host (31)(32)(33).In the case of A. vinelandii, however, the heterologous expression of AvNifH with a high FeS content has not been demonstrated in a foreign host like E. coli.The challenge posed by this task has motivated the consideration of two key parameters for the successful heterologous synthesis of this unique metalloenzyme.One of them is the [Fe 4 S 4 ] cluster of AvNifH, the synthesis of which is carried out by NifS/U in the native A. vinelandii host, with NifS acting as a pyridoxal 5′-phosphate-dependent cysteine desulfurase enzyme that provides sulfur to the scaffold protein NifU for the sequential synthesis of [Fe 2 S 2 ] and [Fe 4 S 4 ] clusters.For the heterologous expression of many FeS proteins in E. coli, IsuS/Uthe homologs of NifS/U-have been successfully used to supplement the endogenous FeS assembly pathways in the expression host and bolster the FeS contents of the heterologously expressed proteins (34)(35)(36)(37).The other parameter is the protein scaffold of AvNifH, the proper folding of which requires NifM, a protein with a partial sequence similarity to known peptidyl-prolyl isomerases and therefore, presumed to enable a cis-trans isomerization of the conserved prolines in AvNifH to facilitate its assembly.Given these considerations, it can be rationalized that co-expression of AvNIfH with AvIscS/U and AvNifM could result in the heterologous synthesis of a functional, FeS-cluster replete form of AvNifH in E. coli.
Here, we report the successful synthesis of a fully active AvNifH protein upon co-expression of this protein with AvIscS/U and AvNifM in E. coli.Our metal, activity, electron paramagnetic resonance (EPR), and XAS (X-ray absorption spectroscopy)/EXAFS data demonstrate a high occupancy of [Fe 4 S 4 ] clusters and a full spectrum of catalytic and biosynthetic activities of the heterologously expressed AvNifH protein.Moreover, our phylogenetic analyses and structural predictions lead to the proposal that AvNifM functions as a chaperone that assists the maturation of a cluster-replete AvNifH protein.
As such, this work provides a useful platform for developing an expression system for the heterologous synthesis of a complete nitrogenase while supplying an effective tool for further investigating the biosynthetic mechanism of this important metalloenzyme.

RESULTS AND DISCUSSION
Co-expression of AvNifH with AvIscS/U and AvNifM in E. coli strain BL21(DE3) resulted in a brown, soluble protein (designated AvNifH Ec ) that was purified at a yield of ∼100 mg protein per 35 g wet cells.A homodimer comprising subunits of ~30 kDa (Fig. 2A), the heterologously expressed AvNifH Ec shows a metal content of 3.3 ± 0.2 mol Fe/mol protein, ~85% of the metal content of 3.9 ± 0.3 mol Fe/mol protein for the AvNifH protein isolated from the native A. vinelandii host (Table S1).Given the presence of one [Fe 4 S 4 ] cluster in this protein, the Fe content of AvNifH Ec suggests an occupancy of >80% of the single [Fe 4 S 4 ] cluster-binding site at its dimeric interface (see Fig. 1).Such a cluster assignment of AvNifH Ec also aligns well with its respective activities in CO 2 -reduction (as an independent enzyme), C 2 H 2 -and N 2 -reduction (with AvNifDK as its catalytic partner), and P-and M-cluster maturation (with precursor-containing AvNifDK and AvNifEN as its respective biosynthetic partners), which range from 77% to 104% of those of its native AvNifH counterpart (Fig. 2B; Table S1).The [Fe 4 S 4 ] cluster of AvNifH Ec has the same ability as that of its native AvNifH counterpart (1,2,11,27) to adopt three oxidation states: the oxidized (+2) state, the reduced (+1) state, and the super-reduced, all-ferrous (0) state.Accordingly, like the native AvNifH, AvNifH Ec is EPR-silent in the oxidized state (Fig. 3A, "Ox") but displays a mixture of S = 3/2 (g = 5.9, 4.3) and S = 1/2 (g = 2.04, 1.94, 1.86) perpendicular-mode EPR signals in the reduced state (Fig. 3A, "Red") (11) as well as a g = 16.4 parallel-mode signal in the super-reduced, all-ferrous state (Fig. 3B, "SR") (27).Other than a slightly stronger g = 4.31 feature of its S = 3/2 signal in the reduced state, the EPR features of AvNifH Ec are comparable in magnitude to those of its native AvNifH counterpart, with the intensities of the S = 3/2 (reduced), S = 1/2 (reduced), and g = 16.4 (super-reduced) signals of AvNifH Ec being 149%, 102%, and 109%, respectively, of those of the corresponding features of AvNifH (Fig. 3A and B).
The close resemblance between the AvNifH Ec -and AvNifH-associated clusters is also reflected by a high degree of similarity between the Fe K-edge XAS/EXAFS data of reduced AvNifH Ec and AvNifH, both showing a primary component in the Fourier transform (FT) that requires Fe-S scattering pathways of ~2.3 Å, as well as a smaller secondary feature in the FT that requires two sets of Fe-Fe scatterers: one at 2.5 Å and the other at 2.7 Å (Fig. 4; Tables S2 to S4).There is a minor shift in the Fe K-edge energy of AvNifH Ec (7,118.3eV) from that of AvNifH (7,117.9eV) (Table S2); however, the overall similarity between the Fe K-edge XAS/EXAFS data of AvNifH Ec and AvNifH, along with the similarities among their metal contents, activities, and EPR features, suggests that the cluster environments in these proteins are highly similar.
The absolute requirement of AvNifM for the successful expression of AvNifH in E. coli points to a crucial role of AvNifM in facilitating the maturation of AvNifH into a functional protein.Previous sequence alignment and mutagenic analysis have led to the proposal that AvNifM is a peptidyl-prolyl isomerase (PPIase) involved in the transition between the cisand trans-forms of the peptide bond of the conserved Pro258 residue in AvNifH (38).Yet, Pro258 is nearly 100% conserved in the NifH species from organisms with or without NifM, casting doubt on the proposed PPIase activity of NifM.Interestingly, based on the structural predictions available in the AlphaFold database (39,40), AvNifM not only possesses a C-terminal domain with a PPIase fold but also has an N-terminal domain with striking resemblance to SurA and PrsA, two proteins known to facilitate protein folding (Fig. 5).A periplasmic chaperone found in many Gram-negative bacteria (e.g., E. coli), SurA possesses two PPIase domains but shows no in vivo PPIase activity (41); instead, it is known to facilitate the folding of outer membrane proteins in the periplasm (42,43).Similarly, PrsA is an extracellular foldase that is best studied in certain Gram-positive bacteria (e.g., Bacillus subtilis) and, despite possessing the characteristic PPIase domain, it serves to assist the folding of a wide range of proteins, including pathogenicity factors and cell wall synthesis proteins (44)(45)(46).Given its architectural similarity to SurA and PrsA, it is likely that NifM also functions as a chaperone that is indispensable for the maturation of a soluble, cluster-replete form of NifH.The exact mode-of-action of NifM in this process, however, remains unclear.
It is interesting to note that while certain diazotrophs (e.g., A. vinelandii) require NifM for the maturation of NifH, most Nif (or homolog) possessing organisms do not contain NifM (Fig. 6A).The presence of NifM is largely limited to a subset of Proteobacteria (i.e., Gammaproteobacteria and Betaproteobacteria) and only occasionally observed in Alphaproteobacteria and other phyla (Fig. 6A).Despite the similarity of its C-terminal domain to PPIase, the N-terminal domain of NifM does not seem to be homologous to any known protein.As such, the evolutionary origin of this protein is unknown.Notably, the gene encoding NifM is almost always present alongside those encoding NifZ and NifW, two nitrogenase assembly/accessory proteins (Fig. 6B); moreover, the three nif genes are almost always grouped in the same genomic architecture: nifWZM.Given the participation of NifZ and NifW in the maturation of the catalytic NifDK component of A. vinelandii (47,48), such a conserved genomic arrangement of nifWZM could imply a functional "grouping" of maturation proteins of nitrogenase and thereby offer additional support for the proposed role of NifM as a chaperone for the maturation of NifH.The question of why NifM is specifically required for the expression of certain NifH species, but not others, requires further investigation.and best fits (solid) of AvNifH Ec (blue) as compared to those of its native AvNifH counterpart (black).See Fig. S2 for Fe K-edge absorption spectra, Table S2 for Fe K-edge energies, and Tables S3 and S4 for details of fits.

Conclusion
In this work, we have successfully synthesized a fully active AvNifH with a high cluster occupancy upon co-expression of this protein with AvIscS/U and AvNifM in E. coli.Our biochemical and spectroscopic data confirm the structural and functional competence of this heterologously expressed AvNifH protein, and our phylogenetic analyses and structural predictions suggest a role of AvNifM as a chaperone for the maturation of a cluster-replete form of AvNifH.Given the crucial role of NifH in nitrogenase catalysis and assembly, this work supplies an essential component for piecing together a complete pathway for the heterologous expression of an active nitrogenase, an ongoing effort that involves further optimization of key parameters and, particularly, the FeS supplies in the expression system (49)(50)(51)(52)(53).Such an expression system of the two-component nitroge nase, along with that of its NifH component alone (as reported herein), could prove useful for the future development of biotechnological applications that harness the reducing prowess of nitrogenase for the production of valuable chemical commodities.Moreover, the expression system described in this work provides a useful tool for tackling the impact of maturation factors, such as AvNifM, in the synthesis of a functional AvNifH, as well as the relationship of various Nif proteins and homologs during evolution, which could prove instrumental in decoding the biosynthetic mechanism of nitrogenase while unraveling the evolutionary origins of this important metalloenzyme.

MATERIALS AND METHODS
All the chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and Thermo Fisher Scientific (Waltham, MA), except when noted otherwise.The experiments were performed either in a glove box or on a Schlenk line, under an atmosphere of Ar with an O 2 concentration of <3 ppm.

Strain construction
The genes encoding A. vinelandii NifH and NifM proteins were synthesized, codon-opti mized for E. coli expression, and then cloned into pET-14b and pRSFDuet-1 vectors, respectively (GenScript, Piscataway, NJ).These constructs were then co-transformed along with a plasmid containing iscSUA and hscABfdx genes from A. vinelandii, which encodes a group of proteins responsible for FeS cluster assembly (34)(35)(36)(37), into the E. coli strain BL21(DE3), resulting in a strain (YM565EE) that expresses His-tagged AvNifH (designated AvNifH Ec ) when induced with isopropyl β-D-1-thiogalactopyranoside (IPTG).The plasmid carrying the iscSUA and hscABfdx genes was kindly provided by Prof. Silke Leimkhüler from the University of Potsdam, Germany.

Cell growth and protein purification
In a BIOFLO 415 fermenter (New Brunswick Scientific) operated at 37°C with 200 rpm agitation and 10 L/min airflow, the E. coli strain YM565EE was cultivated in 10 L batches in LB medium (Difco) supplemented with 50 mM MOPS/NaOH (pH 7.4), 25 mM glucose, 2 mM ferric ammonium citrate, 19 mg/L kanamycin, 28 mg/L chloramphenicol, and 75 mg/L ampicillin.When optical density (OD 600 ) reached 0.5, the airflow was termina ted, and the fermenter was purged with N 2 (ultra-high purity) at a rate of 1.5 L/min; additionally, the temperature was lowered to 24°C before 25 mM sodium fumarate, 2 mM cysteine was added to the culture, and the expression of His-tagged AvNifH Ec was induced upon addition of 250 µM IPTG.The protein was expressed for 16 h before the cells were harvested through centrifugation using a Thermo Fisher Scientific Legend XTR centrifuge.Subsequently, the protein was isolated using immobilized metal affinity chromatography using a method adapted from the purification of His-tagged nitroge nase proteins from A. vinelandii (54).

Metal analysis
The Fe contents of AvNifH and AvNifH Ec were analyzed using inductively coupled plasma optical emission spectroscopy (ICP-OES) with an iCAP7000 ICP-OES instrument (Thermo Scientific).The calibration of the instrument was carried out using standard solutions made from dilution of a 1 mg/mL stock solution of elemental Fe (Thermo-Fisher Scientific).The analysis began by combining the protein sample with 100 µL of concentrated sulfuric acid (H 2 SO 4 ) and 100 µL of concentrated nitric acid (HNO 3 ), followed by heating at 250°C for 30 min.This process was repeated until the solution turned colorless.Subsequently, the solution was allowed to cool to room temperature, followed by dilution of the solution to a total volume of 7.5 mL with 2% HNO 3 and determination of the metal content.

Substrate reduction assays
C 2 H 2 -and N 2 -reduction assays were performed at 30°C in 9.5-mL vials fitted with rubber serum stoppers and metal caps (DWK Life Science, Millville, NJ).The C 2 H 2 -reduction assay contained an atmosphere of 0.1 atm C 2 H 2 and 0.9 atm Ar, while the N 2 -reduction assay contained 1.0 atm of N 2 .Each reaction contained 25 mM Tris-HCl (pH 8.0), 2.5 mM ATP, 5.0 mM MgCl 2 , 30 mM creatine phosphate, 0.125 mg of creatine phosphokinase, and 20 mM sodium dithionite (Na 2 S 2 O 4 ) in a total volume of 1 mL.The reaction was initiated by the addition of 2.4 mg of AvNifDK and 0.36 mg of AvNifH or AvNifH Ec , incubated at 30°C for 10 min, quenched with EDTA, and analyzed for product formation.To detect C 2 H 4 as a product of C 2 H 2 -reduction, 250 µL of the headspace was injected into a GC-FID (SRI Instruments, Torrance, CA) equipped with a packed Poropak N column (Restek, Bellefonte, PA).Calibration was achieved by injecting 15 ppm C 2 H 4 gas standard under the same conditions.To detect NH 3 as the product of N 2 -reduction, 100 µL of the reaction was added to an o-phthalaldehyde (OPA) solution that contained, in a total volume of 1 mL, 10 mM OPA and 2.5 mM 2-mercaptomethanol in a 50 mM potassium phosphate buffer (pH 7.8).The mixture was allowed to sit at room temperature for 3 h, followed by measurement using a fluorescence spectrophotometer (RF-5301PC, Shimadzu Co., Ltd., Japan) using an excitation wavelength of 361 nm and an emission wavelength of 423 nm.

P-cluster maturation assays
Each assay contained 25 mM Tris-HCl (pH 8.0), 0.45 mg of P-cluster precursor (i.e., P*-cluster; 2 × [Fe 4 S 4 ]) containing, yet cofactor-deficient NifDK from A. vinelandii nifH-deletion strain DJ1165 (47), 0.5 mg of AvNifH or AvNifH Ec , 20 mM Na 2 S 2 O 4 , 0.8 mM ATP, 1.6 mM MgCl 2 , 10 mM creatine phosphate, 8 units of creatine phosphokinase, and 10 mL of isolated M-clusters (56) in a total volume of 0.9 mL.The reaction was incubated at 30°C for 60 min and subsequently split into triplicates in three 9.5-mL vials, each containing 1.05 mg of NifH, 25 mM Tris-HCl (pH 8.0), 2.5 mM ATP, 5.0 mM MgCl 2 , 30 mM creatine phosphate, 0.125 mg of creatine phosphokinase, and 20 mM Na 2 S 2 O 4 in a total volume of 0.7 mL, and either C 2 H 2 or N 2 in the headspace as the substrate of the C 2 H 2or N 2 -reduction assay.The reaction mixture was then incubated at 30°C for 10 min and analyzed for product formation as described above.

M-cluster maturation assays
Each assay contained 25 mM Tris-HCl (pH 8.0), 0.45 mg of P-cluster containing, yet cofactor-deficient NifDK from A. vinelandii nifB-deletion strain DJ1143 (47), 1.2 mg of AvNifH or AvNifH Ec , 1.0 mg M-cluster precursor (i.e., L-cluster [Fe 8 S 9 C]) containing NifEN from A. vinelandii strain DJ1041 (55), 0.4 mM homocitrate, 0.4 mM Na 2 MoO 4 , 2.4 mM ATP, 4.8 mM MgCl 2 , 30 mM creatine phosphate, 24 units of creatine phosphokinase, and 20 mM Na 2 S 2 O 4 in a total volume of 0.9 mL.The reaction was incubated at 30°C for 60 min and subsequently split into triplicates in three 9.5-mL vials, each containing 1.05 mg of NifH, 25 mM Tris-HCl (pH 8.0), 2.5 mM ATP, 5.0 mM MgCl 2 , 30 mM creatine phosphate, 0.125 mg of creatine phosphokinase, and 20 mM Na 2 S 2 O 4 in a total volume of 0.7 mL, and either C 2 H 2 or N 2 in the headspace as the substrate of C 2 H 2 -or N 2 -reduc tion assay.The reaction mixture was then incubated at 30°C for 10 min and analyzed for product formation as described above.

EPR analysis
EPR samples were assembled and flash frozen in liquid nitrogenase in a glove box (Vacuum Atmospheres) filled with Ar and maintained at an O 2 concentration of <3 ppm.Three types of samples were prepared for EPR analysis as follows: (i) reduced (Red) samples, which contained 10% (vol/vol) glycerol, 250 mM imidazole, 2 mM Na 2 S 2 O 4 , and 25 mM Tris-HCl (pH 8.0); (ii) oxidized (Ox) samples, which were prepared by incubating the reduced samples with excess indigo disulfonate for 5 min; and (iii) the super-reduced (SR) samples, which were prepared by adding excess europium (II) ethylene glycol-bis(βaminoethyl ether)-N,N,N′,N′-tetraacetic acid (Eu-EGTA), followed by removal of Eu-EGTA on a G25 desalting column (Sigma-Aldrich).
EPR spectra were recorded by an ESP 300E spectrometer (Bruker) interfaced with an ESR-9002 liquid-helium continuous-flow cryostat (Oxford Instruments).The measure ments were taken using a microwave power of 50 mW, a gain factor of 5 × 10 4 , a modulation frequency of 100 kHz, and a modulation amplitude of 5 G. Five scans of perpendicular-mode EPR spectra were recorded for each sample at 10 K (reduced samples) or 15 K (oxidized samples) using a microwave frequency of 9.62 GHz; and eight scans of parallel-mode EPR spectra were recorded for each sample at 10 K (super reduced samples) using a microwave frequency of 9.38 GHz.

XAS spectra
Fe K-edge, XAS spectra were collected with a SPEAR3 storage ring current of ~500 mA and an energy level of 3.0 GeV at two SSRL (Stanford Synchrotron Radiation Lightsource) beamlines: beamline 7-3, which uses a 30-element solid-state Ge detector (Canberra) and beamline 9-3, which uses a 100-element Ge monolith solid-state detector (Canberra).For the Fe scans, an Fe foil was positioned in the beam path before the ionization chamber (I 0 ) and scanned simultaneously for energy calibration, with the first inflection point of the edge assigned to 7,112.0 eV.The potential occurrence of photoreduction was closely monitored by scanning the same spot on the sample twice, which allowed for a comparison between the first derivative peaks associated with the edge energy during the data acquisition process.
The detector channels from the scans were inspected, calibrated, and averaged using the software EXAFSPAK (23).Subsequently, the data were processed for EXAFS analysis with PYSPLINE (57) to obtain χ(k).PYSPLINE was employed to subtract a second-order background across the entire data range and to create a spline function that modeled the background absorption throughout the EXAFS region.A four-region spline was selected, utilizing polynomials of orders 2, 3, and 3 over the post-edge region, and the data were normalized to obtain an edge jump of 1.0 at 7,130 eV.For a specific absorberscatterer pair, theoretical phase and amplitude parameters were computed using FEFF 8.40 (58).Parameters for each species were calculated based on a suitable model derived from the crystal structure of the [Fe 4 S 4 ] cluster in NifH (PDB code 1G5P) (59).
In every analysis conducted, the coordination number of a specific shell (N) was kept as a fixed parameter and changed iteratively in integer steps, while the bond lengths (R) and mean-square deviation (σ²) were permitted to vary without restriction.The estimated uncertainties in R, σ², and N are 0.02 Å, 0.1 × 10⁻³ Å², and 20%, respectively.For the Fe K-edge data, the amplitude reduction factor S ₀ was set at 1.0, while the edge-shift parameter ΔE ₀ was allowed to vary as a singular value across all shells.Therefore, in any particular fit, the number of variable parameters typically equaled two times the number of shells plus 1.The goodness of fit parameters were determined in accordance with the following formulae: Analysis of the pre-edge was carried out on the Fe K-edge fluorescence data, which was normalized to achieve an edge jump of 1.0 at 7,130 eV in PYSPLINE.The features of the pre-edge were fit within the energy range of 7,108 to 7,117 eV using methods detailed elsewhere (60,61).The fitting was conducted using the Fityk program (62), with pseudo-Voigt functions comprising a mixture of Gaussian and Lorentzian functions at a 50:50 ratio.

AlphaFold models
The structural information of AvNifM was retrieved from the AlphaFold Protein Structure Database for entry P14890 (39,63).The predicted structure of the N-terminal domain of AvNifM was then submitted to NCBI VAST (64), which resulted in a hit for the crystal structure of a truncated SurA (PDB code 2PV3) (65) and, consequently, a comparative structural analysis between AvNifM and the full-length SurA structure (PDB code 1M5Y) (66).Further analysis of the predicted structure of AvNifM using FoldSeek (67) led to the identification of PrsA (PDB code 6VJ4) as another close structural homolog of AvNifM (68).PyMOL was used to facilitate structural analysis and visualization (69).

Phylogenetic analysis
The NifH sequences were retrieved from InterPro family IPR005977 (containing AvNifH) (70) and filtered through Reference Proteomes from UniProt (71) to decrease the total number of sequences, which resulted in the identification of 1,502 NifH homolog sequences from 943 organisms.The phylogenetic tree of the organisms was generated based on NCBI taxonomy with phyloT (database version 2022.3)(72) and visualized and annotated in iTOL version 6.7.3 (73).To investigate the presence or the absence of the residue corresponding to Pro258 of AvNifH, the NifH sequences were aligned with MAFFT (74) using automatic settings, and the alignment was trimmed with trimAl (75).The presence or the absence of NifM was investigated by retrieving NifM sequences from InterPro family IPR014282 (containing AvNifM) and matching the sequences by the taxon IDs of the organisms.Similarly, the presence or the absence of NifZ was investigated by retrieving NifZ sequences from InterPro family IPR007415 (containing AvNifZ).The identified NifZ sequences were then submitted to EFI-Genome Neighborhood Tool (76,77), and the NifW sequences neighboring NifZ were annotated accordingly.In cases where more than one NifZ homolog was present in a given organism, the organism was annotated as containing NifW if at least one of the NifZ homologs had a neighboring NifW sequence.

FIG 1
FIG 1 Roles of the Fe protein in substrate reduction and metallocluster assembly.Shown are the functions of Fe protein (NifH) as (A) an electron donor for its catalytic partner, MoFe protein (NifDK), in nitrogenase catalysis; (B) an independent reductase for the reduction of CO or CO 2 into hydrocarbons (HCs); (C) an insertase of Mo/homocitrate (hc) for the maturation of an L-cluster (i.e., M-cluster precursor [Fe 8 S 9 C]) into a fully assembled M-cluster ([(R-homoci trate)MoFe 7 S 9 C]) on a biosynthetic scaffold, NifEN; and (D) a maturase for the reductive coupling of a P*-cluster (i.e., P-cluster precursor; an [Fe 4 S 4 ]-like cluster pair) into a P-cluster ([Fe 8 S 7 ]).All functions of the Fe protein require an electron source (A-D) and, while its functions in nitrogenase catalysis (A) and biosynthesis (C and D) also rely on ATP hydrolysis, its function as an independent reductase (B) can be accomplished with or without MgATP.For the purpose of simplicity, only one αβ-dimer is shown for the tetrameric NifDK in (A).The atoms of the metalloclusters are colored as follows: Fe, orange; S, yellow; Mo, cyan; C, light gray; Mg, green; O, red; Al, dark gray; F, light blue; and P, dark orange.

FIG 2
FIG 2 Biochemical and catalytic analyses of AvNifH Ec .(A) SDS-PAGE of the heterologously expressed AvNifH Ec .(B) Specific activities of C 2 H 2 -reduction (to C 2 H 4 ; light gray), N 2 -reduction (to NH 3 ; dark blue), CO 2 -reduction (to CO; dark red), M-cluster maturation (dark gray), and P-cluster maturation (dark green) by AvNifH Ec as compared to those by its native AvNifH counterpart.Shown above the bars are the activities expressed in percentages, with the activities of AvNifH set as 100% and the activities of AvNifH Ec calculated relative to those of AvNifH (see Table S1 for details).The specific activities are normalized based on the Fe contents of AvNifH Ec (3.3 ± 0.2 mol Fe/mol protein) and AvNifH (3.9 ± 0.3 mol Fe/mol protein).The data points shown in panel (B) represent biological replicates (n = 3) and are expressed as mean ± standard deviation.

FIG 3
FIG 3 EPR analyses of AvNifH Ec .(A) Perpendicular-and (B) parallel-mode EPR spectra of indigo disulfonate-oxidized (Ox), dithionite-reduced (Red), and super-reduced (SR) forms of AvNifH Ec (blue) as compared to those of its native AvNifH counterpart (black).The signal intensities of the EPR spectra are normalized based on the Fe contents of AvNifH Ec (3.3 ± 0.2 mol Fe/mol protein) and AvNifH (3.9 ± 0.3 mol Fe/mol protein).The percentages of the signal intensities of AvNifH Ec as compared to those of AvNifH (set at 100%) are indicated below the EPR traces.Note the characteristic shallow, broad S = 3/2 signals observed in the spectra of both AvNifH and AvNifH Ec in the dithionite-reduced state, which are shown at 10-fold enhanced intensities in Fig. S1.

FIG 4
FIG 4 Fe K-edge XAS analysis of AvNifH Ec .Shown are the Fourier-transformed (A) and k 3 -weighted (B) EXAFS data (dotted)

FIG 5 FIG 6
FIG 5 Structural comparison of AvNifM with SurA and PrsA.(A) AlphaFold structural prediction of AvNifM.Model confidence: blue, very high; cyan, confident; yellow, low; and orange, very low.(B and C) Overlay of the predicted N-terminal (left) and C-terminal (right) domains of AvNifM with those of (B) SurA (PDB: 1M5Y) or (C) PrsA (PDB: 6VJ4).The domains of NifM, SurA, and PrsA are colored blue, light brown, and dark brown, respectively.