Resonance Raman studies of the iron(II)--alpha-keto acid chromophore in model and enzyme complexes.

The bidentate coordination of an alpha-keto acid to an iron(II) center via the keto group and the carboxylate gives rise to metal-to-ligand charge-transfer transitions between 400 and 600 nm in model complexes and in alpha-ketoglutarate-dependent dioxygenases. Excitation into these absorption bands of the Fe(II)TauD(alpha-KG) complex (TauD = taurine/alpha-ketoglutarate dioxygenase, alpha-KG = alpha-ketoglutarate) elicits two resonance Raman features at 460 and 1686 cm(-1), both of which are sensitive to (18)O labeling. Corresponding studies of model complexes, the six-coordinate [Fe(II)(6-Me(3)-TPA)(alpha-keto acid)](+) and the five-coordinate [Fe(II)(Tp(Ph2))(alpha-keto acid)] (6-Me(3)-TPA = tris[(6-methyl-2-pyridyl)methyl]amine, Tp(Ph2) = hydrotris(3,5-diphenylpyrazol-1-yl)borate), lead to the assignment of these two features to the Fe(II)(alpha-keto acid) chelate mode and the nu(C==O) of the keto carbonyl group, respectively. Furthermore, the chelate mode is sensitive to the coordination number of the metal center; binding of a sixth ligand to the five-coordinate [Fe(II)(Tp(Ph2))(benzoylformate)] elicits a 9--20 cm(-1) downshift. Thus, the 10 cm(-1) upshift of the chelate mode observed for Fe(II)TauD(alpha-KG) upon the addition of the substrate, taurine, is associated with the conversion of the six-coordinate metal center to a five-coordinate center, as observed for the iron center of clavaminate synthase from X-ray crystallography (Zhang, Z.; et al. Nat. Struct. Biol. 2000, 7, 127-133) and MCD studies (Zhou, J.; et al. J. Am. Chem. Soc. 1998, 120, 13539--13540). These studies provide useful insights into the initial steps of the oxygen activation mechanism of alpha-ketoglutarate-dependent dioxygenases.

MgATP-Bound and nucleotide-free structures of a nitrogenase protein complex between the Leu 127 Delta-Fe-protein and the MoFe-protein.

A mutant form of the nitrogenase iron protein with a deletion of residue Leu 127, located in the switch II region of the nucleotide binding site, forms a tight, inactive complex with the nitrogenase molybdenum iron (MoFe) protein in the absence of nucleotide. The structure of this complex generated with proteins from Azotobacter vinelandii (designated the L127Delta-Av2-Av1 complex) has been crystallographically determined in the absence of nucleotide at 2.2 A resolution and with bound MgATP (introduced by soaking) at 3.0 A resolution. As observed in the structure of the complex between the wild-type A. vinelandii nitrogenase proteins stabilized with ADP.AlF(4-), the most significant conformational changes in the L127Delta complex occur in the Fe-protein component. While the interactions at the interface between the MoFe-protein and Fe-proteins are conserved in the two complexes, significant differences are evident at the subunit-subunit interface of the dimeric Fe-proteins, with the L127Delta-Av2 structure having a more open conformation than the wild-type Av2 in the complex stabilized by ADP.AlF(4-). Addition of MgATP to the L127Delta-Av2-Av1 complex results in a further increase in the separation between Fe-protein subunits so that the structure more closely resembles that of the wild-type, nucleotide-free, uncomplexed Fe-protein, rather than the Fe-protein conformation in the ADP.AlF(4-) complex. The L127Delta mutation precludes key interactions between the Fe-protein and nucleotide, especially, but not exclusively, in the region corresponding to the switch II region of G-proteins, where the deletion constrains Gly 128 and Asp 129 from forming hydrogen bonds to the gamma-phosphate and activating water for attack on this group, respectively. These alterations account for the inability of this mutant to support mechanistically productive ATP hydrolysis. The ability of the L127Delta-Av2-Av1 complex to bind MgATP demonstrates that dissociation of the nitrogenase complex is not required for nucleotide binding.

ADP-Ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase.

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The structure of the dinitrogenase reductase from Azotobacter vinelandii is known. In this study, mutant forms of dinitrogenase reductase from A. vinelandii that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum. R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction. Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe(4)S(4) cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT. Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT. Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073-2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.

Nitrogenase reduction of carbon disulfide: freeze-quench EPR and ENDOR evidence for three sequential intermediates with cluster-bound carbon moieties.

Freeze-quenching of nitrogenase during reduction of carbon disulfide (CS(2)) was previously shown to result in the appearance of a novel EPR signal (g = 2.21, 1.99, and 1.97) not previously associated with any of the oxidation states of the nitrogenase metal clusters. In the present work, freeze-quench X- and Q-band EPR and Q-band (13)C electron nuclear double resonance (ENDOR) spectroscopic studies of nitrogenase during CS(2) reduction disclose the sequential formation of three distinct intermediates with a carbon-containing fragment of CS(2) bound to a metal cluster inferred to be the molybdenum-iron cofactor. Modeling of the Q-band (35 GHz) EPR spectrum of freeze-trapped samples of nitrogenase during turnover with CS(2) allowed assignment of three signals designated "a" (g = 2.035, 1.982, 1.973), "b" (g = 2.111, 2.002, and 1.956), and "c" (g = 2.211, 1. 996, and 1.978). Freezing samples at varying times after initiation of the reaction reveals that signals "a", "b", and "c" appear and disappear in sequential order. Signal "a" reaches a maximal intensity at 25 s; signal "b" achieves maximal intensity at 60 s; and signal "c" shows maximal intensity at 100 s. To characterize the intermediates, (13)CS(2) was used as a substrate, and freeze-trapped turnover samples were examined by Q-band (13)C ENDOR spectroscopy. Each EPR signal ("a", "b", and "c") gave rise to a distinct (13)C signal, with hyperfine coupling constants of 4.9 MHz for (13)C(a), 1. 8 MHz for (13)C(b), and 2.7 MHz for (13)C(c). Models for the sequential formation of intermediates during nitrogenase reduction of CS(2) are discussed.

Hydrolysis of nucleoside triphosphates other than ATP by nitrogenase.

The hydrolysis of ATP to ADP and P(i) is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron (Fe) protein and the molybdenum-iron (MoFe) protein. The oxidation state of nitrogenase was found to greatly influence the nucleotide hydrolysis rates. MgATP hydrolysis rates were 20 times higher under dithionite reducing conditions (approximately 4,000 nmol of MgADP formed per min/mg of Fe protein) as compared with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed per min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis rates were significantly higher under oxidizing conditions (1,400-2,000 nmol of MgNDP formed per min/mg of Fe protein) as compared with reducing conditions (80-230 nmol of MgNDP formed per min/mg of Fe protein). The K(m) values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330-540 microM) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF(4)(-) resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF(4)(-) x MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.

Stopped-flow kinetic analysis of Escherichia coli taurine/alpha-ketoglutarate dioxygenase: interactions with alpha-ketoglutarate, taurine, and oxygen.

Taurine/alpha-ketoglutarate dioxygenase (TauD), a member of the broad class of non-heme Fe(II) oxygenases, converts taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde while decomposing alpha-ketoglutarate (alphaKG) to form succinate and CO(2). Under anaerobic conditions, the addition of alphaKG to Fe(II)TauD results in the formation of a broad absorption centered at 530 nm. On the basis of studies of other members of the alphaKG-dependent dioxygenase superfamily, we attribute this spectrum to metal chelation by the substrate C-1 carboxylate and C-2 carbonyl groups. Subsequent addition of taurine perturbs the spectrum to yield a 28% greater intensity, an absorption maximum at 520 nm, and distinct shoulders at 480 and 570 nm. This spectral change is specific to taurine and does not occur when 2-aminoethylphosphonate or N-phenyltaurine is added. Titration studies demonstrate that each TauD subunit binds a single molecule of Fe(II), alphaKG, and taurine. In addition, these studies indicate that the affinity of TauD for alphaKG is enhanced by the presence of taurine. alpha-Ketoadipate, the other alpha-keto acid previously shown to support TauD activity, and alpha-ketocaproate lead to the formation of weak 520 nm-like spectra with Fe(II)TauD in the presence of taurine; however, corresponding spectra at 530 nm are not observed in the absence of taurine. Pyruvate and alpha-ketoisovalerate fail to elicit absorption bands in this region of the spectrum, even in the presence of taurine. Stopped-flow UV-visible spectroscopy reveals that the 530 and 520 nm spectra associated with alphaKG-Fe(II)TauD and taurine-alphaKG-Fe(II)TauD are formed at catalytically competent rates ( approximately 40 s(-)(1)). The rate of chromophore formation was independent of substrate or enzyme concentration, suggesting that alphaKG binds to Fe(II)TauD prior to the formation of a chromophoric species. Significantly, the taurine-alphaKG-Fe(II)TauD state, but not the alphaKG-Fe(II)TauD species, reacts rapidly with oxygen (42 +/- 9 s(-)(1)). Using the data described herein, we develop a preliminary kinetic model for TauD catalysis.

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.

In vitro biosynthesis of iron-molybdenum cofactor and maturation of the nif-encoded apodinitrogenase. Effect of substitution for NifH with site-specifically altered forms of NifH.

NifH has three different roles in the nitrogenase enzyme system. Apart from serving as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (apodinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required for the aforementioned processes have been investigated by the use of site-specifically altered forms of the enzyme. The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127Delta, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH to bind and not hydrolyze MgATP is required for it to function in these processes. We also present evidence that the ability of NifH to function in these processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase. Evidence toward the existence of separate, overlapping sites on NifH for each of its functions (substrate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is presented.

Evidence that MgATP accelerates primary electron transfer in a Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein nitrogenase tight complex.

The nitrogenase catalytic cycle involves binding of the iron (Fe) protein to the molybdenum-iron (MoFe) protein, transfer of a single electron from the Fe protein to the MoFe protein concomitant with the hydrolysis of at least two MgATP molecules, followed by dissociation of the two proteins. Earlier studies found that combining the Fe protein isolated from the bacterium Clostridium pasteurianum with the MoFe protein isolated from the bacterium Azotobacter vinelandii resulted in an inactive, nondissociating Fe protein-MoFe protein complex. In the present work, it is demonstrated that primary electron transfer occurs within this nitrogenase tight complex in the absence of MgATP (apparent first-order rate constant k = 0.007 s-1) and that MgATP accelerates this electron transfer reaction by more than 10,000-fold to rates comparable to those observed within homologous nitrogenase complexes (k = 100 s-1). Electron transfer reactions were confirmed by EPR spectroscopy. Finally, the midpoint potentials (Em) for the Fe protein [4Fe-4S]2+/+ cluster and the MoFe protein P2+/N cluster were determined for both the uncomplexed and complexed proteins and with or without MgADP. Calculations from electron transfer theory indicate that the measured changes in Em are not likely to be sufficient to account for the observed nucleotide-dependent rate accelerations for electron transfer.

The [4Fe-4S] cluster domain of the nitrogenase iron protein facilitates conformational changes required for the cooperative binding of two nucleotides.

MgATP binding and hydrolysis are central to all reduction reactions catalyzed by nitrogenase. The iron (Fe) protein component of nitrogenase is a homodimeric protein with a bridging [4Fe-4S] cluster and two nucleotide binding sites, one on each subunit. This work presents evidence that the [4Fe-4S] cluster domain of the nitrogenase Fe protein functions as a hinge region between the two nucleotide binding domains, participating in the cooperative binding of two nucleotides. Alanine residues at position 98 (located near the [4Fe-4S] cluster) of the Azotobacter vinelandii Fe protein were changed by means of site-directed mutagenesis to Val (V) and Gly (G), and the resulting altered proteins were purified and characterized. While the wild-type and A98G Fe proteins were found to bind two nucleotides (MgATP or MgADP) with strong cooperativity (Hill coefficient of 2), the A98V Fe protein was found to bind one nucleotide with no apparent cooperativity. The binding of two nucleotides to the wild-type Fe protein is known to induce protein conformational changes which are reflected as changes in the properties of the [4Fe-4S] cluster, including a change in the redox potential of the [4Fe-4S] cluster of -120 mV for MgATP binding (-300 to -420 mV) and of -160 mV for MgADP binding (-300 to -460 mV). The binding of one nucleotide to the A98V Fe protein was found to result in only half the lowering of the redox potential, with MgATP binding resulting in a -80 mV change (-280 to -360 mV) and MgADP binding resulting in a -50 mV change (-280 to -330 mV). Results from 1H NMR, EPR, and CD spectra, along with Fe chelation rates, were all consistent with the binding of a single nucleotide to the A98V Fe protein inducing a partial conformational change. Finally, the A98V Fe protein with one nucleotide bound, still bound to the molybdenum-iron protein but did not support MgATP hydrolysis, electron transfer, or substrate reduction. A model is discussed in which the [4Fe-4S] cluster domain can be viewed as a hinge region between the two nucleotide binding domains which facilitates conformational rearrangements required for the cooperative binding of a second nucleotide.

Elucidating the mechanism of nucleotide-dependent changes in the redox potential of the [4Fe-4S] cluster in nitrogenase iron protein: the role of phenylalanine 135.

Nucleotide binding to the nitrogenase iron (Fe) protein results in a lowering of the redox potential of its [4Fe-4S] cluster by over 100 mV, and this is thought to be essential for electron transfer to the molybdenum-iron (MoFe) protein for substrate reduction. This work presents evidence for an important role of the strictly conserved phenylalanine at position 135, located near the [4Fe-4S] cluster of nitrogenase Fe protein, in defining both the redox potential and the nucleotide-induced changes in the redox potential of the [4Fe-4S] cluster. Phe 135 was changed by means of site-directed mutagenesis to the amino acids Tyr (F135Y), Ile (F135I), Trp (F135W), and His (F135H), and the altered proteins were purified to homogeneity. Minor changes in the UV/visible and EPR spectra arising from the [4Fe-4S] cluster were detected in the altered proteins, while dramatic changes were observed in the visible region circular dichroism (CD) spectrum, suggesting that Phe 135 contributes significantly to the chiroptical properties of the [4Fe-4S] cluster. Likewise, significant changes in the redox potentials of the Phe altered Fe proteins were observed, with shifts of +50 to +120 mV compared to the redox potential of the wild-type Fe protein (-300 mV). The shifts in redox potential for the altered Fe proteins appeared to correlate with changes in isotropically shifted proton NMR resonances assigned to cluster ligands. All of the Phe 135 altered Fe proteins were found to bind either MgADP or MgATP, while the reduced and oxidized states of the F135W and F135H altered Fe proteins had significantly higher affinities for binding MgATP when compared to the wild-type Fe protein. While MgATP binding to the wild-type and Phe 135 altered Fe proteins resulted in approximately -100 mV shifts in the redox potentials for all proteins, MgADP binding resulted in only -30 to -50 mV shifts for the altered proteins compared to a -160 mV shift for the wild-type Fe protein. The current results suggest that Phe 135 is important in defining the redox potential of the [4Fe-4S] cluster in the Fe protein and influences the MgADP (but not MgATP) induced modulation of the redox potential.

Elucidation of a MgATP signal transduction pathway in the nitrogenase iron protein: formation of a conformation resembling the MgATP-bound state by protein engineering.

The present work defines one MgATP signal transduction pathway in the nitrogenase iron (Fe) protein. Deletion of an amino acid (Leu 127) by site-directed mutagenesis in the protein chain between Asp 125, located in the ATP binding site, and Cys 132, a ligand to the [4Fe-4S] cluster, resulted in protein conformational changes resembling the MgATP-bound state in the absence of any bound nucleotides. Specifically, 1H nuclear magnetic resonance, electron paramagnetic resonance, and circular dichroism spectroscopic properties, along with Fe chelation assays, suggested that deletion of Leu 127 in the Fe protein resulted in changes in the electronic properties of the [4Fe-4S] cluster similar to those normally observed upon MgATP binding to the wild-type Fe protein. Deletion of Leu 127 of the Fe protein lowered the redox potential of of the [4FE-4S] cluster by 112 mV compared to the wild-typeFe protein (-412mV compared to -294 mV). A nearly identical lowering of the redox potential by 120 mV occurs in the wild-type Fe protein upon binding MgATP (-294 mV compared to 420 mV). The L127delta Fe protein did not contain bound nucleotides which could account for the observed conformational changes. The present results support a model in which the protein chain from ASP 125 to Cys 132 acts as one pathway for MgATP signal transduction and suggests a mechanism for this transduction to the [4Fe-4S] cluster. The L127delta Fe protein was found to still bind 2 MgATP or 2 MgADP molecules/Fe protein. Unlike the wild-type Fe protein, the L127delta Fe protein bound 2 ADP molecules/Fe protein in the absence of Mg2+. Finally, the L127delta protein was found to bind to the MoFe protein, although the complex did not catalyze MgATP hydrolysis or substrate reduction. In concurrence with previous models, homologies between the Asp 125 to Cys 132 transduction pathway in Fe protein and the switch II region of the broad class of GTPase signal transduction proteins (G-proteins) are discussed.

Circular dichroism and x-ray spectroscopies of Azotobacter vinelandii nitrogenase iron protein. MgATP and MgADP induced protein conformational changes affecting the [4Fe-4S] cluster and characterization of a [2Fe-2S] form.

Nucleotide interactions with nitrogenase are a central part of the mechanism of nitrogen reduction. Previous studies have suggested that MgATP or MgADP binding to the nitrogenase iron protein (Fe protein) induce protein conformational changes that control component protein docking, interprotein electron transfer, and substrate reduction. In the present study, we have investigated the effects of MgATP or MgADP binding to the Azotobacter vinelandii nitrogenase Fe protein on the properties of the [4Fe-4S] cluster using circular dichroism (CD) and x-ray absorption spectroscopies. Previous CD and magnetic CD studies on nitrogenase Fe protein suggested that binding of either MgATP or MgADP to the Fe protein resulted in identical changes in the CD spectrum arising from transitions of the [4Fe-4S]2+ cluster. We present evidence that MgADP or MgATP binding to the oxidized nitrogenase Fe protein results in distinctly different CD spectra, suggesting distinct changes in the environment of the [4Fc-4S] cluster. The present results are consistent with previous studies such as chelation assays, electron paramagnetic resonance, and NMR, which suggested that MgADP or MgATP binding to the nitrogenase Fe protein induced different conformational changes. The CD spectrum of a [2Fe-2S]2+ form of the nitrogenase Fe protein was also investigated to address the possibility that the MgATP- or MgADP-induced changes in the CD spectrum of the native enzyme were the result of a partial conversion from a [4Fe-4S] cluster to a [2Fe-2S] cluster. No evidence was found for a contribution of a [2Fe-2S]2+ cluster to the CD spectrum of oxidized Fe protein in the absence or presence of nucleotides. A novel two-electron reduction of the [2Fe-2S]2+ cluster in Fe protein was apparent from absorption, CD, and electron paramagnetic resonance data. Fe K-edge x-ray absorption spectra of the oxidized Fe protein revealed no changes in the structure of the [4Fe-4S] cluster upon MgATP binding to the Fe protein. The present results reveal that MgATP or MgADP binding to the oxidized state of the Fe protein result in different conformational changes in the environment around the [4Fe-4S] cluster.

Nucleotide hydrolysis and protein conformational changes in Azotobacter vinelandii nitrogenase iron protein: defining the function of aspartate 129.

The biological reduction of dinitrogen catalyzed by nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron transfer and substrate reduction. The aspartic acid residue at position 129 in the Azotobacter vinelandii Fe protein has been suggested to participate in nucleotide interactions from its location in the X-ray structure near several amino acids previously identified to participate in nucleotide binding and protein conformational changes. The function of this amino acid was probed by changing aspartic acid to glutamic acid (D129E) and asparagine (D129N) by site-directed mutagenesis. The D129N Fe protein proved to be unstable and could not be purified. Characterization of the purified D129E Fe protein revealed a central role for Asp 129 in the nucleotide-induced protein conformational changes in the Fe protein and possibly in the mechanism of MgATP hydrolysis. Data from EPR, circular dichroism spectroscopy, and Fe2+ chelation rates and the chemical shifts of isotropically shifted protons in the 1H NMR spectra implicate Asp 129 in the nucleotide-induced conformational changes in the Fe protein, which are reflected in changes in the environment of the [4Fe-4S] cluster. The D129E Fe protein was found to bind both MgATP and MgADP with high affinity. The Kd determined for MgADP binding (Kd = 131 microM) was comparable to that found for wild-type Fe protein (128 microM). The affinity for MgATP binding was 1.6 times tighter than that for wild-type Fe protein (370 compared to 580 microM). The midpoint reduction potential of the [4Fe-4S] cluster was similar to that determined for the wild-type Fe protein (-290 mV for wild-type Fe protein and -300 mV for D129E Fe protein). Upon the addition of MgATP or MgADP, the midpoint potentials for wild-type and D129E Fe proteins shifted to -430 and -440 mV, respectively. The D129E Fe protein was also found to bind to the molybdenum-iron protein (MoFe protein) with normal affinity, although it could not support electron transfer to the MoFe protein or MoFe protein-stimulated MgATP hydrolysis.

Evidence for a central role of lysine 15 of Azotobacter vinelandii nitrogenase iron protein in nucleotide binding and protein conformational changes.

Biological nitrogen fixation catalyzed by purified nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. In the present study, we demonstrate a central function for Lys-15 of Azotobacter vinelandii nitrogenase iron protein (FeP) in the interaction of nucleotides with nitrogenase. Changing Lys-15 of the FeP to Arg resulted in an FeP with a dramatically reduced affinity for both MgATP and MgADP. From equilibrium column binding experiments at different nucleotide concentrations, apparent dissociation constants (Kd) for wild type FeP binding of MgADP (143 microM) and MgATP (571 microM) were determined. Over the same nucleotide concentration ranges, the K15R FeP showed no significant affinity for either nucleotide. This contrasts sharply with previous results with an FeP in which Lys-15 was changed to Gln (K15Q) where it was found that the K15Q FeP bound MgADP with the same affinity as wild type FeP and MgATP with a slightly reduced affinity. Analysis of K15R FeP by EPR, circular dichroism (CD), and microcoulometry revealed that the [4Fe-4S] cluster was unaffected by the amino acid change and that addition of either MgADP or MgATP did not result in the protein conformational changes normally detected by these techniques. These results are integrated into a model for how MgATP and MgADP bind and induce conformational changes within the FeP.