MoFe Protein Research Articles

The genome of Paenibacillus sabinae T27 provides insight into evolution, organization and functional elucidation of nif and nif-like genes.

BackgroundMost biological nitrogen fixation is catalyzed by the molybdenum nitrogenase. This enzyme is a complex which contains the MoFe protein encoded by nifDK and the Fe protein encoded by nifH. In addition to nifHDK, nifHDK-like genes were found in some Archaea and Firmicutes, but their function is unclear.ResultsWe sequenced the genome of Paenibacillus sabinae T27. A total of 4,793 open reading frames were predicted from its 5.27 Mb genome. The genome of P. sabinae T27 contains fifteen nitrogen fixation (nif) genes, including three nifH, one nifD, one nifK, four nifB, two nifE, two nifN, one nifX and one nifV. Of the 15 nif genes, eight nif genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX and nifV) and two non-nif genes (orf1 and hesA) form a complete nif gene cluster. In addition to the nif genes, there are nitrogenase-like genes, including two nifH-like genes and five pairs of nifDK-like genes. IS elements on the flanking regions of nif and nif-like genes imply that these genes might have been obtained by horizontal gene transfer. Phylogenies of the concatenated 8 nif gene (nifB, nifH, nifD, nifK, nifE, nifN, nifX and nifV) products suggest that P. sabinae T27 is closely related to Frankia. RT-PCR analysis showed that the complete nif gene cluster is organized as an operon. We demonstrated that the complete nif gene cluster under the control of σ70-dependent promoter enabled Escherichia coli JM109 to fix nitrogen. Also, here for the first time we demonstrated that unlike nif genes, the transcriptions of nifHDK-like genes were not regulated by ammonium and oxygen, and nifH-like or nifD-like gene could not restore the nitrogenase activity of Klebsiella pneumonia nifH− and nifD− mutant strains, respectively, suggesting that nifHDK-like genes were not involved in nitrogen fixation.ConclusionsOur data and analysis reveal the contents and distribution of nif and nif-like genes and contribute to the study of evolutionary history of nitrogen fixation in Paenibacillus. For the first time we demonstrated that the transcriptions of nifHDK-like genes were not regulated by ammonium and oxygen and nifHDK-like genes were not involved in nitrogen fixation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-723) contains supplementary material, which is available to authorized users.

Reconstitution of a sequential reaction of two nitrogenase-like enzymes in the bacteriochlorophyll biosynthetic pathway of Rhodobacter capsulatus

The parental structure of bacteriochlorophyll a, bacteriochlorin, is formed by a sequential operation of two nitrogenase-like enzymes, dark-operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide a oxidoreductase (COR). Both DPOR and COR consist of two components, Fe protein and MoFe protein cognates. Here we determined kinetic parameters of COR and established the reconstitution system for the formation of bacteriochlorin (3-vinyl bacteriochlorophyllide a) from porphyrin (protochlorophyllide) with purified components of DPOR and COR from Rhodobacter capsulatus. This reconstitution system confirmed the recent finding that COR catalyzes 8-vinyl reduction of 8-vinyl chlorophyllide a in addition to the known activity of C7C8 double bond reduction, and provides a promising model to investigate how two nitrogenase-like enzymes are coordinated in bacteriochlorophyll biosynthesis.

Substrate Channel in Nitrogenase Revealed by a Molecular Dynamics Approach

Mo-dependent nitrogenase catalyzes the biological reduction of N2 to two NH3 molecules at FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized submicrosecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel. The viability of this observed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, resulting in the discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment and that approaches a face of FeMo-cofactor earlier implicated in substrate binding.

A confirmation of the quench-cryoannealing relaxation protocol for identifying reduction states of freeze-trapped nitrogenase intermediates.

We have advanced a mechanism for nitrogenase catalysis that rests on the identification of a low-spin EPR signal (S = 1/2) trapped during turnover of a MoFe protein as the E4 state, which has accumulated four reducing equivalents as two [Fe–H–Fe] bridging hydrides. Because electrons are delivered to the MoFe protein one at a time, with the rate-limiting step being the off-rate of oxidized Fe protein, it is difficult to directly control, or know, the degree of reduction, n, of a trapped intermediate, denoted En, n = 1–8. To overcome this previously intractable problem, we introduced a quench-cryoannealing relaxation protocol for determining n of an EPR-active trapped En turnover state. The trapped “hydride” state was allowed to relax to the resting E0 state in frozen medium, which prevents additional accumulation of reducing equivalents; binding of reduced Fe protein and release of oxidized protein from the MoFe protein both are abolished in a frozen solid. Relaxation of En was monitored by periodic EPR analysis at cryogenic temperature. The protocol rests on the hypothesis that an intermediate trapped in the frozen solid can relax toward the resting state only by the release of a stable reduction product from FeMo-co. In turnover under Ar, the only product that can be released is H2, which carries two reducing equivalents. This hypothesis implicitly predicts that states that have accumulated an odd number of electrons/protons (n = 1, 3) during turnover under Ar cannot relax to E0: E3 can relax to E1, but E1 cannot relax to E0 in the frozen state. The present experiments confirm this prediction and, thus, the quench-cryoannealing protocol and our assignment of E4, the foundation of the proposed mechanism for nitrogenase catalysis. This study further gives insights into the identity of the En intermediates with high-spin EPR signals, 1b and 1c, trapped under high electron flux.

Nitrogenase FeMo cofactor: an atomic structure in three simple steps

The enzyme nitrogenase catalyzes the six-electron reduction of molecular dinitrogen to ammonium, concomitant with the reduction of protons to yield hydrogen gas. In the MoFe protein component of the nitrogenase system, the unique FeMo cofactor is the active site of catalysis, but its exact mechanism remains under debate. This review focuses on the history of the structure determination of FeMo cofactor, a process that extended over two decades and involved several iterations and corrections that are unusual and unexpected within the established field of X-ray crystallography. However, FeMo cofactor has defied expectations on several occasions, and besides being the largest single iron-sulfur cluster known to bioinorganic chemistry, this [Mo:7Fe:9S:C]:homocitrate moiety is unique in some aspects that have misled researchers several times. We have now arrived at a final and complete description of the atomic structure of FeMo cofactor, and yet many questions regarding various aspects of the catalytic mechanism of this enzyme remain to be answered.

Nonenzymatic Synthesis of the P-Cluster in the Nitrogenase MoFe Protein: Evidence of the Involvement of All-Ferrous [Fe4S4]0 Intermediates

The P-cluster in the nitrogenase MoFe protein is a [Fe8S7] cluster and represents the most complex FeS cluster found in Nature. To date, the exact mechanism of the in vivo synthesis of the P-cluster remains unclear. What is known is that the precursor to the P-cluster is a pair of neighboring [Fe4S4]-like clusters found on the ΔnifH MoFe protein, a protein expressed in the absence of the nitrogenase Fe protein (NifH). Moreover, incubation of the ΔnifH MoFe protein with NifH and MgATP results in the synthesis of the MoFe protein P-clusters. To improve our understanding of the mechanism of this reaction, we conducted a magnetic circular dichroism (MCD) spectroscopic study of the [Fe4S4]-like clusters on the ΔnifH MoFe protein. Reducing the ΔnifH MoFe protein with Ti(III) citrate results in the quenching of the S = 1/2 electron paramagnetic resonance signal associated with the [Fe4S4]+ state of the clusters. MCD spectroscopy reveals this reduction results in all four 4Fe clusters being converted into the unusual, all-ferrous [Fe4S4]0 state. Subsequent increases of the redox potential generate new clusters. Most significantly, one of these newly formed clusters is the P-cluster, which represents approximately 20–25% of the converted Fe concentration. The other two clusters are an X cluster, of unknown structure, and a classic [Fe4S4] cluster, which represents approximately 30–35% of the Fe concentration. Diamagnetic FeS clusters may also have been generated but, because of their low spectral intensity, would not have been identified. These results demonstrate that the nitrogenase P-cluster can be generated in the absence of NifH and MgATP.

Mutagenesis at α-423Ile of MoFe protein reduces the catalytic activity of nitrogenase in Klebsiella oxytoca

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia, the central process of biological nitrogen fixation. The mechanism of electron transfer from the P-cluster to the iron-molybdenum cofactor (FeMo-co) during the process of substrate reduction remains unclear. Based on our previous hypothesis, the dual pathway model, three mutants of Klebsiella oxytoca strain M5al (termed Nβ102A, Kα431H and Iα423P) were constructed to map the H+/e− transfer pathway, in which β-102Asn, α-431Lys and α-423Ile of the molybdenum-iron (MoFe) protein of nitrogenases were replaced by alanine, histidine and proline, respectively. All three substitutions affected the diazotrophic growth, especially of the Iα423P strain with very low capability of diazotrophic growth and C2H2 reduction activity. In a 42-L fermenter, the Iα423P strain was induced to the maximal C2H2 reduction activity, which was only ~17 % of that of M5al. However, the transcription level of the nifD gene was approximately four-fold higher than that of the M5al strain, indicating that the low C2H2 reduction activity of Iα423P was not because of downregulation of nifD expression. The maximal C2H2 and H+ reduction activities of the Iα423P MoFe protein were 13 % and 21 % of those of the wild-type MoFe protein, respectively. In conclusion, we propose that the changed properties of the Iα423P MoFe protein are related to the disruption of hydrogen bond formation between α-423Ile and homocitrate, and that α-423Ile serves as a gate to the Mo site in FeMo-co during the H+/e− transfer in nitrogenase.

A Substrate Channel in Nitrogenase Revealed by a Molecular Dynamics Approach

Mo-dependent nitrogenase catalyzes the biological reduction of N2 to 2NH3 at the FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized sub-microsecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel not previously reported. The viability of the proposed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, with discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment that approaches a face of FeMo-cofactor earlier implicated in substrate binding.

Biosynthesis of Nitrogenase Metalloclusters

Biosynthesis of Nitrogenase Metalloclusters

Functional divergence outlines the evolution of novel protein function in NifH/BchL protein family

Biological nitrogen fixation is accomplished by prokaryotes through the catalytic action of complex metalloenzyme, nitrogenase. Nitrogenase is a two-protein component system comprising MoFe protein (NifD and K) and Fe protein (NifH). NifH shares structural and mechanistic similarities as well as evolutionary relationships with light-independent protochlorophyllide reductase (BchL), a photosynthesis-related metalloenzyme belonging to the same protein family. We performed a comprehensive bioinformatics analysis of the NifH/BchL family in order to elucidate the intrinsic functional diversity and the underlying evolutionary mechanism among the members. To analyse functional divergence in the NifH/ BchL family, we have conducted pair-wise estimation in altered evolutionary rates between the member proteins. We identified a number of vital amino acid sites which contribute to predicted functional diversity. We have also made use of the maximum likelihood tests for detection of positive selection at the amino acid level followed by the structure-based phylogenetic approach to draw conclusion on the ancient lineage and novel characterization of the NifH/BchL protein family. Our investigation provides ample support to the fact that NifH protein and BchL share robust structural similarities and have probably deviated from a common ancestor followed by divergence in functional properties possibly due to gene duplication.

The Sixteenth Iron in the Nitrogenase MoFe Protein

Noch ein Eisen im Feuer: Anomale Röntgenbeugungsanalysen der MoFe-Nitrogenase decken die Gegenwart eines einkernigen Eisenzentrums auf, bezeichnet als Fe16, das vormals als entweder Ca2+ oder Mg2+ identifiziert worden war. Die Lage der Absorptionskante verweist auf einen Oxidationszustand dieses Zentrums von +2. Die hohe Sequenzkonservierung der an Fe16 koordinierten Aminosäurereste unterstreicht die potenzielle Bedeutung dieses Zentrums für die Nitrogenasefunktion.

The Sixteenth Iron in the Nitrogenase MoFe Protein

Another iron in the fire: X-ray anomalous diffraction studies on the nitrogenase MoFe protein show the presence of a mononuclear iron site, designated as Fe16, which was previously identified as either Ca(2+) or Mg(2+). The position of the absorption edge indicates that this site is in the oxidation state +2. The high sequence conservation of the residues coordinated to Fe16 emphasizes the potential importance of the site in nitrogenase.

Reduction of Fluorinated Cyclopropene by Nitrogenase

Reduction of the first known halogen-containing substrate by nitrogenase (N2ase), 3,3-difluorocyclopropene (DFCP), was investigated. Reduction requires both N2ase proteins (MoFe and Fe protein), ATP, and an exogenous reductant (dithionite, DT), as with N2 and known alternative substrates of the enzyme. Two major products providing evidence for reductive C-F bond cleavage were confirmed, propene (P1, requiring 6e(-)/6H(+)) and 2-fluoropropene (P2, 4e(-)/4H(+)). Both were identified by GC-MS and NMR spectroscopy, and had the same Km constants (0.022 atm, 5.4 mM). Reduction of 1,2-dideuterated DFCP (d2-DFCP) further revealed that (i) in both P1 and P2, two deuterium atoms are retained, one on carbon-1 and one on carbon-3, indicating that C═C bond cleavage rather than C-C bond cleavage is involved during DFCP reduction at least to P2 (assuming no F migration); (ii) no selectivity was observed in formation of cis and trans isomers of 1,3-d2-2-fluoropropene, whereas cis-1,3-d2-propene is the predominant 1,3-d2-propene product, indicating that one of the bound reduction intermediates on the pathway to propene is constrained geometrically. A reduction mechanism, consistent with hydride transfer as a key step, is discussed. Reductive C-F bond cleavage is an ability of N2ase that further demonstrates the unique and remarkable scope of its catalytic prowess.

Nucleotide-Assisted [Fe4S4] Redox State Interconversions of the Azotobacter vinelandii Fe Protein and Their Relevance to Nitrogenase Catalysis

The [Fe4S4] cluster of the nitrogenase Fe protein from Azotobacter vinelandii can exist in three redox states: oxidized [Fe4S4](2+), dithionite reduced [Fe4S4](1+), and two forms of the all ferrous [Fe4S4](0), S = 4 and 0. Operation of the [Fe4S4](2+)/ [Fe4S4](1+) redox couple transfers one electron to the MoFe protein during catalysis with hydrolysis of two MgATPs. In contrast, the [Fe4S4](2+)/[Fe4S4](0) couple transfers two electrons per binding event, accompanied by hydrolysis of only two MgATPs. Both reactions occur at nearly identical rates even though the number of electrons transferred differs by a factor of 2. MgATP and MgADP facilitate interconversion of the three redox states: 2[Fe4S4](1+) + 4 MgATP = [Fe4S4](2+)(MgATP)2 + [Fe4S4](0)(MgATP)2, as demonstrated by the MgATP reaction. This reaction was investigated as a possible precursor reaction to provide two electrons in the form of [Fe4S4](0)(MgATP)2 for delivery to the MoFe protein to then conduct a two-electron substrate reduction. However, experiments showed that this disproportionation reaction, which readily occurs, was not viable during nitrogenase catalysis utilizing the [Fe4S4](1+) cluster state. The known cooperative behavior of the Fe protein in the [Fe4S4](1+) state taken together with a measured turnover potential of -460 mV with an n = 2 value, suggest a gating process on the MoFe protein involving a two electron step.

ADP‐ribosylation, a mechanism regulating nitrogenase activity

Nitrogen fixation is the vital biochemical process in which atmospheric molecular nitrogen is made available to the biosphere. The process is highly energetically costly and thus tightly regulated. The activity of the key enzyme, nitrogenase, is controlled by reversible mono-ADP-ribosylation of one of its components, the Fe protein. This protein provides the other component, the MoFe protein, with the electrons required for the reduction of molecular nitrogen. The Fe-protein is ADP-ribosylated and de-ADP-ribosylated by dinitrogenase reductase ADP-ribosyl transferase and dinitrogenase reductase activating glycohydrolase, respectively. Here we review the current biochemical and structural knowledge of this central regulatory reaction.

THE MOLECULAR ENZYMOLOGY OF NITROGEN FIXATION

ABSTRACT The requirements for ATP and reductant during diazotrophic growth, the physicochemical properties of the nitrogenase proteins, and the involvement of Mo in different aspects of N2 fixation are reviewed. Irrespective of the source from which it is isolated, nitrogenase has very similar properties and is comprised of two O2-sensitive redox proteins. The Mo—Fe protein is an α2β2 tetramer of MW 220000, containing 2 Mo and approx. 33 ± 3 Fe and acid-labile S atoms per tetramer. The Mo atoms and approx. 40% of the Fe atoms can be extracted with N-methylformamide as a unique cofactor (FeMoco) which contains Mo, Fe and S2- in the ratios 1:8:4. Cluster extrusion and Mossbauer data are consistent with the presence of four 4Fe4S clusters in addition to two FeMoco clusters. The binding of substrate (C2H2), its reduction product C2H4, and the inhibitor CO to the Mo-Fe protein have been investigated by EPR spectroscopy. The Fe protein is γ2 dimer of MW 67000; spectroscopic and extrusion data indicate the prese...

THE PRESENT STATUS OF HYDROGEN RECYCLING IN LEGUMES

ABSTRACT A brief discussion is presented of recent information concerning (a) factors influencing extent of N2, loss during N2 fixation by legumes; (b) electron carriers involved in the oxyhydrogen reaction of Rhizobium japonicum bacteroids; and (c) progress made in evaluating H2 recycling advantages. A major factor determining whether H2 is evolved from legume nodules is the presence of an active uptake hydrogenase which participates in the oxidation of H2 that is evolved as a by-product of the nitrogenase reaction. The extent of H2 evolution from the nitrogenase reaction is affected by those factors that influence the nitrogenase turnover rate. These include the supply of ATP and reductant and the ratio of the Fe protein to the MoFe protein component of nitrogenase. Oxidation of H2 in Rhizobium bacteroids is catalyzed by a series of enzymes located in bacteroid membranes. In addition to the hydrogenase per se, carriers so far shown to be involved in the process include cytochromes of the b and c types a...

Nitrogenase: A Draft Mechanism

Biological nitrogen fixation, the reduction of N(2) to two NH(3) molecules, supports more than half the human population. The predominant form of the enzyme nitrogenase, which catalyzes this reaction, comprises an electron-delivery Fe protein and a catalytic MoFe protein. Although nitrogenase has been studied extensively, the catalytic mechanism has remained unknown. At a minimum, a mechanism must identify and characterize each intermediate formed during catalysis and embed these intermediates within a kinetic framework that explains their dynamic interconversion. The Lowe-Thorneley (LT) model describes nitrogenase kinetics and provides rate constants for transformations among intermediates (denoted E(n), where n is the number of electrons (and protons), that have accumulated within the MoFe protein). Until recently, however, research on purified nitrogenase had not characterized any E(n) state beyond E(0). In this Account, we summarize the recent characterization of three freeze-trapped intermediate states formed during nitrogenase catalysis and place them within the LT kinetic scheme. First we discuss the key E(4) state, which is primed for N(2) binding and reduction and which we refer to as the "Janus intermediate" because it lies halfway through the reaction cycle. This state has accumulated four reducing equivalents stored as two [Fe-H-Fe] bridging hydrides bound to the active-site iron-molybdenum cofactor ([7Fe-9S-Mo-C-homocitrate]; FeMo-co) at its resting oxidation level. The other two trapped intermediates contain reduced forms of N(2). One, intermediate, designated I, has S = 1/2 FeMo-co. Electron nuclear double resonance/hyperfine sublevel correlation (ENDOR/HYSCORE) measurements indicate that I is the final catalytic state, E(8), with NH(3) product bound to FeMo-co at its resting redox level. The other characterized intermediate, designated H, has integer-spin FeMo-co (non-Kramers; S ≥ 2). Electron spin echo envelope modulation (ESEEM) measurements indicate that H contains the [-NH(2)] fragment bound to FeMo-co and therefore corresponds to E(7). These assignments in the context of previous studies imply a pathway in which (i) N(2) binds at E(4) with liberation of H(2), (ii) N(2) is promptly reduced to N(2)H(2), (iii) the two N's are reduced in two steps to form hydrazine-bound FeMo-co, and (iv) two NH(3) are liberated in two further steps of reduction. This proposal identifies nitrogenase as following a "prompt-alternating (P-A)" reaction pathway and unifies the catalytic pathway with the LT kinetic framework. However, the proposal does not incorporate one of the most puzzling aspects of nitrogenase catalysis: obligatory generation of H(2) upon N(2) binding that apparently "wastes" two reducing equivalents and thus 25% of the total energy supplied by the hydrolysis of ATP. Because E(4) stores its four accumulated reducing equivalents as two bridging hydrides, we propose an answer to this puzzle based on the organometallic chemistry of hydrides and dihydrogen. We propose that H(2) release upon N(2) binding involves reductive elimination of two hydrides to yield N(2) bound to doubly reduced FeMo-co. Delivery of the two available electrons and two activating protons yields cofactor-bound diazene, in agreement with the P-A scheme. This keystone completes a draft mechanism for nitrogenase that both organizes the vast body of data on which it is founded and serves as a basis for future experiments.

Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

A doubly substituted form of the nitrogenase MoFe protein (α-70(Val)(→Ala), α-195(His→Gln)) has the capacity to catalyze the reduction of carbon dioxide (CO(2)) to yield methane (CH(4)). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH(4) within 20 min. The catalytic rate depends on the partial pressure of CO(2) (or concentration of HCO(3)(-)) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H(2)C = CH-CH(3)) through the reductive coupling of CO(2) and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO(2) sequestration and formation of olefins.

The Nitrogenase Regulatory Enzyme Dinitrogenase Reductase ADP-Ribosyltransferase (DraT) Is Activated by Direct Interaction with the Signal Transduction Protein GlnB

Fe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy in Azospirillum brasilense, Rhodospirillum rubrum, and Rhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling P(II) proteins. In A. brasilense, two P(II) proteins, GlnB and GlnZ, were identified. We used Fe protein from Azotobacter vinelandii as the substrate to assess the activity of A. brasilense DraT in vitro complexed or not with P(II) proteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The P(II) effector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated P(II) proteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that the A. brasilense DraT-GlnB complex was at least 18-fold more efficient than DraT purified from R. rubrum, but with a similar K(m) value for NAD(+). Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.