Dinitrogenase Reductase-activating Glycohydrolase Research Articles

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.

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzyme's preference for manganese over magnesium, although not requiring a redox active metal for the reaction.

Crystal Structure of Dinitrogenase Reductase-activating Glycohydrolase (DRAG) Reveals Conservation in the ADP-Ribosylhydrolase Fold and Specific Features in the ADP-Ribose-binding Pocket

Protein-reversible ADP-ribosylation is emerging as an important post-translational modification used to control enzymatic and protein activity in different biological systems. This modification regulates nitrogenase activity in several nitrogen-fixing bacterial species. ADP-ribosylation is catalyzed by ADP-ribosyltransferases and is reversed by ADP-ribosylhydrolases. The structure of the ADP-ribosylhydrolase that acts on Azospirillum brasilense nitrogenase (dinitrogenase reductase-activating glycohydrolase, DraG) has been solved at a resolution of 2.5 Å. This bacterial member of the ADP-ribosylhydrolase family acts specifically towards a mono-ADP-ribosylated substrate. The protein shows an all-α-helix structure with two magnesium ions located in the active site. Comparison of the DraG structure with orthologues deposited in the Protein Data Bank from Archaea and mammals indicates that the ADP-ribosylhydrolase fold is conserved in all domains of life. Modeling of the binding of the substrate ADP-ribosyl moiety to DraG is in excellent agreement with biochemical data.

In Vitro Interactions between the PII Proteins and the Nitrogenase Regulatory Enzymes Dinitrogenase Reductase ADP-ribosyltransferase (DraT) and Dinitrogenase Reductase-activating Glycohydrolase (DraG) in Azospirillum brasilense

The activity of the nitrogenase enzyme in the diazotroph Azospirillum brasilense is reversibly inactivated by ammonium through ADP-ribosylation of the nitrogenase NifH subunit. This process is catalyzed by DraT and is reversed by DraG, and the activities of both enzymes are regulated according to the levels of ammonium through direct interactions with the P(II) proteins GlnB and GlnZ. We have previously shown that DraG interacts with GlnZ both in vivo and in vitro and that DraT interacts with GlnB in vivo. We have now characterized the influence of P(II) uridylylation status and the P(II) effectors (ATP, ADP, and 2-oxoglutarate) on the in vitro formation of DraT-GlnB and DraG-GlnZ complexes. We observed that both interactions are maximized when P(II) proteins are de-uridylylated and when ADP is present. The DraT-GlnB complex formed in vivo was purified to homogeneity in the presence of ADP. The stoichiometry of the DraT-GlnB complex was determined by three independent approaches, all of which indicated a 1:1 stoichiometry (DraT monomer:GlnB trimer). Our results suggest that the intracellular fluctuation of the P(II) ligands ATP, ADP, and 2-oxoglutarate play a key role in the post-translational regulation of nitrogenase activity.

Formation of a cross-linking complex of dinitrogenase reductase-activating glycohydrolase (DRAG) with membrane proteins from Rhodospirillum rubrum chromatophores

Association of dinitrogenase reductase-activating glycohydrolase (DRAG) with membrane proteins of chromatophores has been investigated. The formation of a multicomponent complex between DRAG and membrane proteins was demonstrated in the presence of glutaraldehyde and EDC/NHS (N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride/hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid sodium salt). Complex formation was observed both in native chromatophore membrane and in chromatophores treated with 0.5 M NaCl in the presence of homogeneous DRAG and glutaraldehyde in cross-reaction. The molecular weight of the complex was around 200 kD, which is consistent with the association of DRAG with three or more chromatophore membrane proteins. A specific complex with molecular weight of about 75 kD was formed only in the presence of EDC/NHS in the cross-linking reaction. It was demonstrated that ammonium transport protein and P11 protein are possible candidates for association with DRAG in chromatophore membranes.

Identification of Rhodospirillum rubrum GlnB Variants That Are Altered in Their Ability To Interact with Different Targets in Response to Nitrogen Status Signals

In Rhodospirillum rubrum, NifA, the transcriptional activator for the nif genes, is posttranslationally activated only by the uridylylated form of GlnB, one of three P(II) homologs in the organism. We have used the yeast two-hybrid system to detect variants of GlnB that interact better with NifA than does wild-type GlnB. When examined for physiological effects in R. rubrum, these GlnB* variants activated NifA in the presence of NH(4)(+), which normally blocks NifA activation completely, and in the absence of GlnD, whose uridylylation of GlnB is also normally essential for NifA activation. When these variants were tested in the two-hybrid system for their interaction with NtrB, a receptor that should interact with the nonuridylylated form of GlnB, they were uniformly weaker than wild-type GlnB in that interaction. When expressed in R. rubrum either as single-copy integrants or on multiple-copy plasmids, these variants were also dramatically altered in terms of their ability to regulate several other receptors involved in nitrogen metabolism, including GlnE, NtrB/NtrC, and DRAT (dinitrogenase reductase ADP-ribosyl transferase)-DRAG (dinitrogenase reductase-activating glycohydrolase). The consistent pattern throughout is that these GlnB variants partially mimic the uridylylated form of wild-type GlnB, even under nitrogen-excess conditions and in strains lacking GlnD. The results suggest that the role of uridylylation of GlnB is primarily to shift the equilibrium of GlnB from a "nitrogen-sufficient" form to a "nitrogen-deficient" form, each of which interacts with different but overlapping receptor proteins in the cell. These GlnB variants apparently shift that equilibrium through direct structural changes.

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.

Characterization of altered regulation variants of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum

In Rhodospirillum rubrum, nitrogenase activity is subject to posttranslational regulation through the adenosine diphosphate (ADP)-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG). To study the posttranslational regulation of DRAG, its gene was mutagenized and colonies screened for altered DRAG regulation. Three different mutants were found and the DRAG variants displayed different biochemical properties including an altered affinity for divalent metal ions. Taken together, the results suggest that the site involved in regulation is physically near the metal binding site of DRAG.

Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status.

The GlnB (P(II)) protein, the product of glnB, has been characterized previously in the photosynthetic bacterium Rhodospirillum rubrum. Here we describe identification of two other P(II) homologs in this organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell. While GlnB is required for activation of NifA activity in R. rubrum, GlnK and GlnJ do not appear to be involved in this process. In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of dinitrogenase reductase catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase-activating glycohydrolase (DRAG) regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.

Effects of specific amino acid substitutions on activities of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum.

Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

Regulation of nitrogenase in the photosynthetic bacteriumRhodobacter sphaeroidescontainingdraTGandnifHDKgenes fromRhodobacter capsulatus

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.Key words: nitrogenase regulation, nitrogenase modification, photosynthetic bacteria.

Regulation of nitrogenase in the photosynthetic bacterium <i>Rhodobacter sphaeroides</i> containing <i>draTG</i> and <i>nifHDK</i> genes from <i>Rhodobacter capsulatus</i>

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.

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.

Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum.

Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH(4)(+) and darkness treatments.

Regulation of Dinitrogenase Reductase ADP-ribosyltransferase and Dinitrogenase Reductase-activating Glycohydrolase by a Redox-dependent Conformational Change of Nitrogenase Fe Protein

The nitrogenase-regulating enzymes dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG), from Rhodospirillum rubrum, were shown to be sensitive to the redox status of the [Fe(4)S(4)](1+/2+) cluster of nitrogenase Fe protein from R. rubrum or Azotobacter vinelandii. DRAG had <2% activity with oxidized R. rubrum Fe protein relative to activity with reduced Fe protein. The activity of DRAG with oxygen-denatured Fe protein or a low molecular weight substrate, N(alpha)-dansyl-N(omega)-(1,N(6)-etheno-ADP-ribosyl)-arginine methyl ester, was independent of redox potential. The redox midpoint potential of DRAG activation of Fe protein was -430 mV versus standard hydrogen electrode, coinciding with the midpoint potential of the [Fe(4)S(4)] cluster from R. rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low (<20%) activity with 87% reduced R. rubrum Fe protein relative to activity with fully oxidized Fe protein. A mutant of R. rubrum in which the rate of oxidation of Fe protein was substantially decreased had a markedly slower rate of ADP-ribosylation in vivo in response to 10 mM NH(4)Cl or darkness stimulus. It is concluded that the redox state of Fe protein plays a significant role in regulation of the activities of DRAT and DRAG in vivo.

Dinitrogenase reductase-activating glycohydrolase can be released from chromatophores of Rhodospirillum rubrum by treatment with MgGDP.

Dinitrogenase reductase-activating glycohydrolase (DRAG), involved in the regulation of nitrogenase activity in Rhodospirillum rubrum, is associated with chromatophore membranes in cell extracts. We show that DRAG can be specifically released by treatment with MgGDP; other nucleotides studied had no effect. The DRAG activity released corresponds to the release of DRAG protein.

Cloning, sequencing and transcriptional regulation of the draT and draG genes of Azospirillum lipoferum FS

From Azospirillum lipoferum (Al) FS, a nitrogen-fixing bacterium isolated from the rhizosphere of rice, we cloned and sequenced draT, encoding dinitrogenase reductase ADP-ribosyltransferase, and draG, encoding dinitrogenase reductase-activating glycohydrolase. The nucleotide sequences of draTG showed extensive similarity to the same genes from Azospirillum brasilense, Rhodospirillum rubrum and Rhodobacter capsulatus, and they are assumed to be co-transcribed as a single operon. When this draTG operon was introduced into Klebsiella oxytoca, this organism acquired the ability to respond to extracellular NH 4 +ions with reversible inhibition of nitrogenase activity, similar to that seen in Al FS. We constructed a plasmid containing a draT::lacZ gene fusion and found that β-galactosidase activity was detected under microaerobic conditions, regardless of NH 4 + concentration, but not under aerobic conditions. This indicates that the transcription of draTG responds to the level of oxygen, but not to that of NH 4 + ions.

Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense

Reversible ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system, has been characterized in both Rhodospirillum rubrum and Azospirillum brasilense. Although the general functions of DRAT and DRAG are very similar in these two organisms, there are a number of interesting differences, e.g., in the timing and extent of the regulatory response to different stimuli. In this work, the basis of these differences has been studied by the heterologous expression of either draTG or nifH from A. brasilense in R. rubrum mutants that lack these genes, as well as the expression of draTG from R. rubrum in an A. brasilense draTG mutant. In general, these hybrid strains respond to stimuli in a manner similar to that of the wild-type parent of the recipient strain rather than the wild-type source of the introduced genes. These results suggest that the differences seen in the regulatory response in these organisms are not primarily a result of different properties of DRAT, DRAG, or dinitrogenase reductase. Instead, the differences are likely the result of different signal pathways that regulate DRAG and DRAT activities in these two organisms. Our results also suggest that draT and draG are cotranscribed in A. brasilense.

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.

Purification and characterization of an oxygen-stable form of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum

Dinitrogenase reductase-activating glycohydrolase (DRAG) is responsible for removing the ADP-ribose moiety from post-translationally inactivated nitrogenase of Rhodospirillum rubrum. Using DRAG purified from an overexpressing strain (UR276), further properties of this enzyme were studied, including its u.v.-visible and fluorescence spectra and its stability in air. DRAG appears to require no covalently bound inorganic cofactors for its activity or regulation. Previously, purified DRAG was found to be rapidly inactivated in air. The air-catalysed lability originated with the presence of sodium dithionite and Mn2+ throughout the purification of the enzyme. This lability can be mimicked using H2O2, which is known to oxidatively inactivate proteins containing bivalent metals. Implications for the regulation of nitrogenase are discussed with respect to the lack of sensitivity to air of the regulatory enzyme, DRAG.