Homocitrate cures the NifV- phenotype in Klebsiella pneumoniae.

Dinitrogenase was isolated from a culture of a Klebsiella pneumoniae NifV- strain derepressed for nitrogenase in the presence of homocitrate. The enzyme isolated from this culture was identical to the wild-type dinitrogenase. These data provide in vivo evidence that the absence of homocitrate is responsible for the NifV- phenotype.     

Dinitrogenase with altered substrate specificity results from the use of homocitrate analogues for in vitro synthesis of the iron-molybdenum cofactor

The in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires homocitrate (2-hydroxy-1,2,4-butanetricarboxylic acid). Homocitrate is apparently synthesized by the nifV gene product. In the absence of homocitrate, no FeMo-co is formed in vitro, as determined from coupled C2H2 reduction assays and the lack of 99Mo label incorporation into apodinitrogenase. Several organic acids were tested for their ability to replace homocitrate in the FeMo-co synthesis system. With appropriate homocitrate analogues, aberrant forms of FeMo-co are synthesized that exhibit altered substrate specificity and inhibitor susceptibility. Homoisocitrate (1-hydroxy-1,2,4-butanetricarboxylic acid) and 2-oxoglutarate facilitated the incorporation of 99Mo into apodinitrogenase in the FeMo-co synthesis system, yielding a dinitrogenase that effectively catalyzed the reduction of protons but not C2H2 or N2. Citrate also promoted the incorporation of 99Mo into apodinitrogenase, and the resulting holodinitrogenase reduced protons and C2H2 effectively but not N2. In addition, proton reduction from this enzyme was inhibited by CO. The properties of the homodinitrogenase formed in the presence of citrate were reminiscent of those of the Klebsiella pneumoniae NifV- dinitrogenase. We also observed low rates of HD formation from NifV- dinitrogenase compared to those from the wild-type enzyme. No HD formation was observed with the dinitrogenase activated in vitro in the presence of citrate. We propose that in vivo NifV- mutants utilize citrate for FeMo-co synthesis.     

Citrate substitutes for homocitrate in nitrogenase of a nifV mutant of Klebsiella pneumoniae

An organic acid extracted from purified dinitrogenase isolated from a nifV mutant of Klebsiella pneumoniae has been identified as citric acid. H2 evolution by the citrate-containing dinitrogenase is partially inhibited by CO, and by some substrates for nitrogenase. The response of maximum velocities to changes in pH for both the wild-type and the NifV- dinitrogenase was compared. No substantial differences between the enzymes were observed, but there are minor differences. Both enzymes are stable in the pH range 4.8-10, but the enzyme activities dropped dramatically below pH 6.2.     

Substrate reduction properties of dinitrogenase activated in vitro are dependent upon the presence of homocitrate or its analogues during iron-molybdenum cofactor synthesis

(R)-2-Hydroxy-1,2,4-butanetricarboxylic acid [(R)-homocitrate] has been has been recently reported to be an integral constituent of the otherwise thought to be inorganic iron-molybdenum cofactor of dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V.K. (1989) Biochemistry 28,2768-2771]. Different organic acids can substitute for homocitrate in an in vitro system for iron-molybdenum cofactor synthesis and incorporation into dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V. K. (1988) Biochemistry 27, 3647-3652]. Dinitrogenase activated with homocitrate-FeMo-co was able to reduce dinitrogen, acetylene, and protons efficiently. Homoisocitrate and isocitrate dinitrogenases did not reduce dinitrogen or acetylene, but showed very high proton reduction activities. Citrate and citramalate dinitrogenases had very low dinitrogen reduction activities and intermediate acetylene and proton reduction activities. CO inhibited proton reduction in both these cases but not in the case of dinitrogenases activated with other homocitrate analogues. By use of these and other commercially available homocitrate analogues in the in vitro system, the structural features of the homocitrate molecule absolutely required for the synthesis of a catalytically competent iron-molybdenum cofactor were determined to be the hydroxyl group, the 1- and 2-carboxyl groups, and the R configuration of the chiral center. The stringency of the structural requirements was dependent on the nitrogenase substrate used for the assay, with dinitrogen having the most stringent requirements followed by acetylene and protons.     

Biosynthesis of the iron-molybdenum cofactor of nitrogenase

The iron-molybdenum cofactor (FeMo-co) of nitrogenase is a Mo-Fe-S cluster that has been proposed as the site of substrate reduction for the nitrogenase enzyme complex. Biosynthesis of FeMo-co in Klebsiella pneumoniae requires at least six nif (nitrogen fixation) gene products. One of the nif genes, nifV, apparently encodes a homocitrate synthase. The synthesis and accumulation of homocitrate [(R)-2-hydroxy-1,2,4-butanetricarboxylic acid] in K.pneumoniae is correlated to the presence of a functional nifV gene. K.pneumoniae strains with mutations in nifV synthesize and accumulate an aberrant form of FeMo-co. Nitrogenase from NifV- mutants is capable of reducing some of the substrates of nitrogenase effectively (e.g. acetylene), but reduces N2 poorly. With the aid of an in vitro FeMo-co synthesis system, it recently has been established that homocitrate is an endogenous component of FeMo-co. Substitution of homocitrate with other carboxylic acids results in the formation of aberrant forms of FeMo-co with altered substrate reduction capability.     

Requirement of homocitrate for the transfer of a 49V-labeled precursor of the iron-vanadium cofactor from VnfX to nif-apodinitrogenase

A vanadium- and iron-containing cluster has been shown previously to accumulate on VnfX in the Azotobacter vinelandii mutant strain CA11.1 (DeltanifHDKvnfDGK::spc). In the present study, we show the homocitrate-dependent transfer of (49)V label from VnfX to nif-apodinitrogenase in vitro. This transfer of radiolabel correlates with acquisition of acetylene reduction activity. Acetylene is reduced both to ethylene and ethane by the hybrid holodinitrogenase so formed, a feature characteristic of alternative nitrogenases. Structural analogues of homocitrate prevent the acetylene reduction ability of the resulting dinitrogenase. Addition of NifB cofactor (-co) or a source of vanadium (Na(3)VO(4) or VCl(3)) does not increase nitrogenase activity. Our results suggest that there is in vitro incorporation of homocitrate into the V-Fe-S cluster associated with VnfX and that the completed cluster can be inserted into nif-apodinitrogenase. The homocitrate incorporation reaction and the insertion of the cluster into nif-apodinitrogenase (alpha(2)beta(2)gamma(2)) do not require MgATP. Attempts to achieve FeV-co synthesis using extracts of other FeV-co-negative mutants were unsuccessful, showing that earlier steps in FeV-co synthesis, such as the steps requiring VnfNE or VnfH, do not occur in vitro.     

Homocitrate is a component of the iron-molybdenum cofactor of nitrogenase

When apodinitrogenase (lacking FeMo-co) was activated with FeMo-co synthesized in vitro in the presence of 3H-labeled homocitrate, label was incorporated into dinitrogenase. The physical association of the label with FeMo-co was demonstrated by reisolation and purification of the cofactor from dinitrogenase. The presence of homocitrate in FeMo-co was established by NMR analysis of the organic acid extracted from dinitrogenase. Quantitation of homocitrate in dinitrogenase showed it to be present at a 1:1 ratio with molybdenum.     

Ammonium inhibition of nitrogenase activity in Herbaspirillum seropedicae.

The effect of oxygen, ammonium ion, and amino acids on nitrogenase activity in the root-associated N2-fixing bacterium Herbaspirillum seropedicae was investigated in comparison with Azospirillum spp. and Rhodospirillum rubrum. H. seropedicae is microaerophilic, and its optimal dissolved oxygen level is from 0.04 to 0.2 kPa for dinitrogen fixation but higher when it is supplied with fixed nitrogen. No nitrogenase activity was detected when the dissolved O2 level corresponded to 4.0 kPa. Ammonium, a product of the nitrogenase reaction, reversibly inhibited nitrogenase activity when added to derepressed cell cultures. However, the inhibition of nitrogenase activity was only partial even with concentrations of ammonium chloride as high as 20 mM. Amides such as glutamine and asparagine partially inhibited nitrogenase activity, but glutamate did not. Nitrogenase in crude extracts prepared from ammonium-inhibited cells showed activity as high as in extracts from N2-fixing cells. The pattern of the dinitrogenase and the dinitrogenase reductase revealed by the immunoblotting technique did not change upon ammonium chloride treatment of cells in vivo. No homologous sequences were detected with the draT-draG probe from Azospirillum lipoferum. There is no clear evidence that ADP-ribosylation of the dinitrogenase reductase is involved in the ammonium inhibition of H. seropedicae. The uncoupler carbonyl cyanide m-chlorophenylhydrazone decreased the intracellular ATP concentration and inhibited the nitrogenase activity of whole cells. The ATP pool was not significantly disturbed when cultures were treated with ammonium in vivo. Possible mechanisms for inhibition by ammonium of whole-cell nitrogenase activity in H. seropedicae are discussed.     

Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.

The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.     

Cross-functionality of nitrogenase components NifH1 and VnfH in Anabaena variabilis

Anabaena variabilis fixes nitrogen under aerobic growth conditions in differentiated cells called heterocysts using either a Mo nitrogenase or a V nitrogenase. The nifH1 gene, which encodes the dinitrogenase reductase of the Mo nitrogenase that is expressed only in heterocysts, is cotranscribed with nifD1 and nifK1, which together encode the Mo dinitrogenase. These genes were expressed in the presence or absence of molybdate or vanadate. The vnfH gene, which encodes the dinitrogenase reductase of the V nitrogenase, was located about 23 kb from vnfDGK, which encodes the V dinitrogenase; however, like vnfDGK, vnfH was expressed only in the absence of molybdate, with or without vanadate. Like nifH1, the vnfH gene was expressed exclusively in heterocysts under either aerobic or anaerobic growth conditions and thus is under the control of developmental factors. The vnfH mutant was able to grow diazotrophically using the V nitrogenase, because NifH1, which was also made in cells starved for molybdate, could substitute for VnfH. Under oxic conditions, the nifH1 mutant grew in the absence of molybdate but not in its presence, using VnfH, while the nifH1 vnfH double mutant did not grow diazotrophically with or without molybdate or vanadate. A nifH1 mutant that expressed nifDK and vnfH but not vnfDGK was able to grow and fix nitrogen normally, indicating that VnfH could substitute for NifH in the Mo nitrogenase and that these dinitrogenase reductases are not involved in determining the metal specificity of the Mo nitrogenase or the V nitrogenase.     

Subcellular localization of the homocitrate synthase in<Emphasis Type="Italic"> Penicillium chrysogenum</Emphasis>

There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.     

Evidence for a dynamic role for homocitrate during nitrogen fixation: the effect of substitution at the alpha-Lys426 position in MoFe-protein of Azotobacter vinelandii

Although it is generally accepted that the active site of nitrogenase is located on the FeMo-cofactor, the exact site(s) of N2 binding and reduction remain the subject of continuing debate, with both molybdenum and iron atoms being suggested as key players. The current consensus favours binding of acetylene and some other non-biologically relevant substrates to the central iron atoms of the FeMo-cofactor [Dos Santos, Igarashi, Lee, Hoffman, Seefeldt and Dean (2005) Acc. Chem. Res. 38, 208-214]. The reduction of N2 is, however, a more demanding process than reduction of these alternative substrates because it has a much higher activation energy and does not bind until three electrons have been accumulated on the enzyme. The possible conversion of bidentate into monodentate homocitrate on this three electron-reduced species has been proposed to free up a binding site for N2 on the molybdenum atom. One of the features of this hypothesis is that alpha-Lys426 facilitates chelate ring opening and subsequent orientation of the monodentate homocitrate by forming a specific hydrogen bond to the homocitrate -CH2CH2CO2- carboxylate group. In support of this concept, we show that mutation of alpha-Lys426 can selectively perturb N2 reduction without affecting acetylene reduction. We interpret our experimental observations in the light of a detailed molecular mechanics modelling study of the wild-type and altered MoFe-nitrogenases.     

Reversible membrane association of dinitrogenase reductase activating glycohydrolase in the regulation of nitrogenase activity in Rhodospirillum rubrum; dependence on GlnJ and AmtB1

In the photosynthetic bacterium Rhodospirillum rubrum nitrogenase activity is regulated by reversible ADP-ribosylation of dinitrogenase reductase in response to external so called "switch-off" effectors. Activation of the modified, inactive form is catalyzed by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the ADP-ribose moiety. This study addresses the signal transduction between external effectors and DRAG. R. rubrum, wild-type and P(II) mutant strains, were studied with respect to DRAG localization. We conclude that GlnJ clearly has an effect on the association of DRAG to the membrane in agreement with the effect on regulation of nitrogenase activity. Furthermore, we have generated a R. rubrum mutant lacking the putative ammonium transporter AmtB1 which was shown not to respond to "switch-off" effectors; no loss of nitrogenase activity and no ADP-ribosylation. Interestingly, DRAG was mainly localized to the cytosol in this mutant. Overall the results support our model in which association to the membrane is part of the mechanism regulating DRAG activity.     

Inhibition of iron-molybdenum cofactor biosynthesis by L127Delta NifH and evidence for a complex formation between L127Delta NifH and NifNE.

Besides serving as the obligate electron donor to dinitrogenase during nitrogenase turnover, dinitrogenase reductase (NifH) is required for the biosynthesis of the iron-molybdenum cofactor (FeMo-co) and for the maturation of alpha(2)beta(2) apo-dinitrogenase (apo-dinitrogenase maturation). In an attempt to understand the role of NifH in FeMo-co biosynthesis, a site-specific altered form of NifH in which leucine at position 127 has been deleted, L127Delta, was employed in in vitro FeMo-co synthesis assays. This altered form of NifH has been shown to inhibit substrate reduction by the wild-type nitrogenase complex, forming a tight protein complex with dinitrogenase. The L127Delta NifH was found to inhibit in vitro FeMo-co synthesis by wild-type NifH as detected by the gamma gel shift assay. Increasing the concentration of NifNE and NifB-cofactor (NifB-co) relieved the inhibition of FeMo-co synthesis by L127Delta NifH. The formation of a complex of L127Delta NifH with NifNE was investigated by gel filtration chromatography. We herein report the formation of a complex between L127Delta NifH and NifNE in the presence of NifB-co. This work presents evidence for one of the possible roles for NifH in FeMo-co biosynthesis, i.e. the interaction of NifH with a NifNE.NifB-co complex.     

Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase.

Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation.     

Effects of perturbations of the nitrogenase electron transfer chain on reversible ADP-ribosylation of nitrogenase Fe protein in Klebsiella pneumoniae strains bearing the Rhodospirillum rubrum dra operon

The redox state of nitrogenase Fe protein is shown to affect regulation of ADP-ribosylation in Klebsiella pneumoniae strains transformed by plasmids carrying dra genes from Rhodospirillum rubrum. The dra operon encodes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase, enzymes responsible for the reversible inactivation, via ADP-ribosylation, of nitrogenase Fe protein in R. rubrum. In bacteria containing the dra operon in their chromosomes, inactivation occurs in response to energy limitation or nitrogen sufficiency. The dra gene products, expressed at a low level in K. pneumoniae, enable transformants to reversibly ADP-ribosylate nitrogenase Fe protein in response to the presence of fixed nitrogen. The activities of both regulatory enzymes are regulated in vivo as described in R. rubrum. Genetic perturbations of the nitrogenase electron transport chain were found to affect the rate of inactivation of Fe protein. Strains lacking the electron donors to Fe protein (NifF or NifJ) were found to inactivate Fe protein more quickly than a strain with wild-type background. Deletion of nifD, which encodes a subunit of nitrogenase MoFe protein, was found to result in a slower inactivation response. No variation was found in the reactivation responses of these strains. It is concluded that the redox state of the Fe protein contributes to the regulation of the ADP-ribosylation of Fe protein.     

Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium.

Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.     

Regulation of Nitrogen Fixation in Klebsiella pneumoniae: Evidence for a Role of Glutamine Synthetase as a Regulator of Nitrogenase Synthesis

Mutations causing constitutive synthesis of glutamine synthetase (GlnC(-) phenotype) were transferred from Klebsiella aerogenes into Klebsiella pneumoniae by P1-mediated transduction. Such GlnC(-) strains of K. pneumoniae have constitutive levels of glutamine synthetase. Two of three GlnC(-) strains of K. pneumoniae studied, each containing independently isolated mutations that confer the GlnC(-) phenotype, continue to synthesize nitrogenase in the presence of NH(4) (+). One strain, KP5069, produces 30% as much nitrogenase when grown in the presence of 15 mM NH(4) (+) as in its absence. The GlnC(-) phenotype allows the synthesis of nitrogenase to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain. Glutamine auxotrophs of K. pneumoniae, that do not produce catalytically active glutamine synthetase, are unable to synthesize nitrogenase during nitrogen limited growth. Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F'133) simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase. These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae.     

Expression of glnB and a glnB-Like Gene (glnK) in a Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Mutant of Rhodobacter sphaeroides

In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia.     

NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum.

Chemical cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum has been investigated with a cross-linking system utilizing two reagents, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and sulfo-N-hydroxysuccinimide. Cross-linking between dinitrogenase reductase and DRAT requires the presence of NAD, the cellular ADP-ribose donor, or a NAD analog containing an unmodified nicotinamide group, such as nicotinamide hypoxanthine dinucleotide. NADP, which will not replace NAD in the modification reaction, does support cross-linking between dinitrogenase reductase and DRAT. The DRAT-catalyzed ADP-ribosylation of dinitrogenase reductase is inhibited by sodium chloride, as is the cross-linking between dinitrogenase reductase and DRAT, suggesting that ionic interactions are required for the association of these two proteins. Cross-linking is specific for native, unmodified dinitrogenase reductase, in that both oxygen-denatured and ADP-ribosylated dinitrogenase reductase fail to form a cross-linked complex with DRAT. The ADP-bound and adenine nucleotide-free states of dinitrogenase reductase form cross-linked complexes with DRAT; however, cross-linking is inhibited when dinitrogenase reductase is in its ATP-bound state.