©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for a Central Role of Lysine 15 of Azotobacter vinelandii Nitrogenase Iron Protein in Nucleotide Binding and Protein Conformational Changes (*)

Matthew J. Ryle (1), William N. Lanzilotta (1), Leonard E. Mortenson (2), Gerald D. Watt (3), Lance C. Seefeldt (1)(§)

From the (1) Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, the (2) Department of Biochemistry, University of Georgia, Athens, Georgia 30602, and the (3) Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Biological nitrogen fixation catalyzed by purified nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N 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 (K ) for wild type FeP binding of MgADP (143 µM) and MgATP (571 µM) 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.


INTRODUCTION

The hydrolysis of a minimum of 16 MgATP to 16 MgADP and 16 P is required for the reduction of each N to 2 NH by purified nitrogenase (for reviews, see Refs. 1-5). The exact mode of nucleotide binding, the mechanism of MgATP hydrolysis, and the mechanism of coupling of this hydrolysis to substrate reduction within nitrogenase remains largely undefined. Nitrogenase is a complex metalloenzyme consisting of two separable metalloproteins, the molybdenum-iron protein (MoFeP) and the iron protein (FeP). The MoFeP contains the site of substrate binding and reduction which is thought be at its molybdenum-iron containing cofactor (FeMoco) (6, 7) . The MoFeP also contains a unique [8Fe-(7-8)S] cluster (8Fe or P cluster) which is proposed to mediate the transfer of electrons from the FeP to FeMoco (6, 8, 9) . The FeP is a homodimeric protein which contains a single [4Fe-4S] cluster bridged between its subunits (10) . The consensus model suggests that the FeP functions to transfer a single electron from its [4Fe-4S] cluster to the MoFeP (probably the 8Fe cluster) along with the concomitant hydrolysis of 2 MgATP for each electron transferred. The resulting oxidized FeP, with 2 MgADP bound, is released from the MoFeP, is reduced by electron carrier proteins and the MgADP is replaced by MgATP (11) . The above cycle is repeated to provide sufficient electrons for substrate reduction on the MoFeP.

While only the FePMoFeP complex will hydrolyze bound MgATP, the FeP alone is known to bind 2 MgATP or 2 MgADP per dimer. The binding of these nucleotides to the FeP induces conformational changes in the FeP which affect the properties of its [4Fe-4S] cluster (4) . Significant among these changes is the reduction of the midpoint potential of the cluster from -310 to -430 mV (12, 13) and the increased accessibility of the cluster to chelators (14, 15) , suggesting greater solvent exposure. These conformational changes have thus been suggested to be a prerequisite for the intercomponent electron transfer between the FeP and the MoFeP. While the nucleotide induced conformational change of the FeP is required for intercomponent electron transfer, it is not sufficient. Rather, the hydrolysis of MgATP appears to be an absolute requirement for electron transfer. The mechanism of coupling of these two processes is complex and probably involves multiple conformational changes in both FeP and MoFeP (16, 17) .

In an attempt to elucidate the mechanism of nucleotide utilization by nitrogenase, we are defining the nucleotide binding site on the FeP, residues involved in hydrolysis, and residues responsible for the conformational change. In recent work, we identified an amino acid sequence in the Azotobacter vinelandii FeP that is involved in nucleotide interaction (18) . This sequence, located near the N terminus of the FeP, is similar to motifs known to function in nucleotide binding in other proteins (i.e. P-loop or Walker A motif) (19) . Specifically, we demonstrated that the highly conserved lysine at position 15 of this motif plays a major role in both the FeP interaction with the -phosphate of MgATP and in the MgATP induced conformational change. We found that changing Lys-15 to the neutral amino acid Gln by site-directed mutagenesis resulted in an FeP that still bound MgADP with normal affinity, but showed a slightly reduced affinity for MgATP binding (18) . In the present work, we extend the function of the A. vinelandii FeP Lys-15 by analysis of a protein in which Lys-15 has been changed to Arg. Analysis of the K15R FeP reveal significant differences in nucleotide interactions when compared to the wild type orK15Q FeP. A model is presented explaining a central function for Lys-15 in nucleotide interactions with the FeP.


MATERIALS AND METHODS

Site-directed Mutagenesis, Expression, and Purification of Altered FeP

Oligonucleotide directed mutagenesis of the A. vinelandii nitrogenase FeP gene, nifH, was carried out as described previously (18, 20) . The mutated nifH gene was incorporated into the A. vinelandii chromosome in place of the wild type nifH gene for expression (20, 21) . Altered FeP was expressed in A. vinelandii cells and purified as described (20) . Protein concentrations were determined by using the Biuret assay (22) with bovine serum albumin as the standard. SDS-polyacrylamide gels were prepared as described (18) and were stained with Coomassie Blue.

MgATP-dependent Chelation of Fefrom Wild Type and K15R FeP

The chelation of Fe from both the wild type and K15R FeP by ,`-dipyridyl was performed as described previously (18) . Spectrophotometric measurements were done on a Hewlett Packard 8452A diode array spectrophotometer.

Equilibrium MgADP and MgATP Binding to Wild Type and K15R FeP

The binding of MgATP or MgADP to the reduced wild type or reduced K15R FeP was determined by a modification of an equilibrium column binding technique (18, 20) . A series of Sephadex G-25 columns (0.7 15 cm) were equilibrated with 50 mM Tris buffer, pH 8.0, containing 2 mM dithionite and various concentrations of either MgADP (0-300 µM) or MgATP (0-1500 µM). A fixed amount of FeP was loaded onto each column and the columns were developed. The protein containing fractions were collected and the concentration of protein was determined by a modified Biuret method (22) and the concentration of nucleotide was quantified by separation and analysis by high performance liquid chromatography (20) . Midpoint Potential of the [4Fe-4S] Cluster in Wild Type and K15R FeP-The midpoint potentials for the [4Fe-4S]/[4Fe-4S] couple in wild type or K15R FeP were determined by microcoulometry as described previously (12, 23). In an argon filled glove box, reduced FeP containing 2 mM NaSO was oxidized by adding an excess of 5.6 mM indigo disulfonate just until a blue color remained. The oxidized FeP (FePox) was separated from small molecules by passage through a Sephadex G-25 column (0.7 10 cm) equilibrated with 50 mM Tris buffer, pH 8, under argon. The concentration of FePox was determined from its absorption spectrum and an absorption coefficient of 13.3 mM cm at 400 nm (24) . Electrochemical reductions were determined for 150-µg aliquots of FePox in 50 mM Tris buffer, pH 8.0, with 200 mM NaCl including 0.1 mM methyl viologen, 0.1 mM benzyl viologen, and 0.1 mM flavin mononucleotide as mediators. For determination of midpoint potentials with nucleotides, either MgATP or MgADP was added to a final concentration of 3 mM. The number of electrons per protein was plotted against the applied potential ranging from -150 mV to -450 mV at 30 mV increments. The data were fit to the Nernst equation to determine the midpoint reduction potential and the number of electrons per protein. All potentials are relative to the standard hydrogen electrode.

EPR

Samples to be analyzed by EPR were frozen in liquid nitrogen as described previously (18) . MgATP was added as a 5-fold molar excess over protein from a stock solution prior to freezing. All spectra were recorded on a Bruker ER200D spectrometer with an Oxford ESR9 cryostat.

Circular Dichroism Spectra of FeP Samples With or Without Nucleotides

FeP samples (wild type or K15R; 550 µl of 69 mgml) were desalted on a Sephadex G-25 column (0.5 10 cm) equilibrated with 100 mM Tris buffer, pH 7.5. The FeP samples were oxidized by the addition of 20 µl of an anaerobic 20 mM indigo disulfonate solution. The oxidized FeP was separated from small molecules by passage through a Sephadex G-25 column (0.5 10 cm) as above. The FePox was diluted to 4 ml with 100 mM Tris buffer, pH 7.5, and 2 ml of this solution was transferred into a 1-cm path length, argon-purged quartz cuvette fitted with a butyl stopper. MgATP or MgADP were added as a 5-fold molar excess over protein from a stock solution. All spectra were recorded using a Jasco model J-500 spectropolarimeter and were baseline corrected.


RESULTS

Lys-15 of FeP

Most nucleotide binding proteins contain a series of conserved amino acids which are involved in nucleotide interaction (19) . The most common of these motifs is the sequence GXXXXGKS/T (Walker A or P-loop) where several of these amino acids have been shown to interact with the phosphate portion of the bound nucleotide (25) . It has been previously shown that nitrogenase FeP from a range of bacteria all contain this conserved nucleotide binding motif near the N terminus (26) . Substitution of the strictly conserved Lys of this sequence in A. vinelandii FeP (Lys-15) with the neutral amino acid Gln resulted in an FeP that was inactive in both electron transfer to the MoFeP and MgATP hydrolysis (18) . Analysis of the K15Q protein suggested that this Lys interacted specifically with the -phosphate of MgATP and not with the - or -phosphates of MgADP and was essential to the nucleotide-induced conformational changes (18) . Subsequent solution of the x-ray crystal structure of the FeP revealed that Lys-15 is located in a loop analogous to the phosphate binding loop in other nucleotide binding proteins and that Lys-15 was located near the phosphates of an adventitiously bound ADP in the structure (10) . The x-ray structure also suggested that Lys-15 might form a salt bridge with Asp-125, which has subsequently been shown to be part of a nucleotide-induced conformational switch (27) . To probe the possible significance of this salt bridge, it was of interest to change Lys-15 to the positively charged amino acid arginine. This substitution would have the effect of maintaining the positive charge, but would change the length of the side chain and the possible hydrogen bonding to Asp-125.

Like the Gln substituted FeP (K15Q), the arginine substituted FeP (K15R) was found to be inactive in electron transfer, MgATP hydrolysis, and substrate reduction. More detailed analysis of the properties of the K15R FeP revealed, however, several differences from the K15Q FeP which suggested a much broader role for Lys-15 in nucleotide interaction.

MgATP-induced Conformational Changes in the K15R FeP

Given the role of Lys-15 in nucleotide interaction, it was of interest to examine the nature of MgATP interactions with K15R FeP. MgATP binding to wild type FeP results in changes in the environment of the [4Fe-4S] cluster which can be monitored by several techniques. The EPR spectrum of the reduced [4Fe-4S] cluster in wild type FeP has a rhombic line shape with signals arising from both spin 1/2 (g = 2 region) and spin 3/2 (g = 5 region) states (28, 29) . Addition of MgATP to the FeP results in a change in both the proportion of spin 1/2 and spin 3/2 signals and in a change in the spin 1/2 signal line shape to axial (Fig. 1). Analysis of the K15R FeP by EPR revealed a normal rhombic EPR spectrum in the absence of nucleotide (Fig. 1), confirming that changing Lys-15 to Arg did not result in conformational changes in the protein that might result in a perturbation of the [4Fe-4S] cluster. Significantly, the addition of MgATP to the K15R FeP did not result in any changes in the EPR spectrum, suggesting a lack of nucleotide-induced conformational changes.


Figure 1: EPR spectra of purified wild type FeP and K15R FeP with and without MgATP. Purified wild type (69 mgml) and K15R (69 mgml) FeP were prepared as described under ``Materials and Methods.'' All samples contained 2 mM dithionite and were prepared and frozen under argon. All spectra were recorded at 4 K, 9.44 GHz, and 15 milliwatts microwave power. Where noted, MgATP was added as a 5-fold molar excess over protein prior to freezing.



MgATP-induced conformational changes in the FeP have also been shown to result in a time-dependent chelation of the Fe from the cluster by iron specific chelators (14, 15) . This has been interpreted to indicate an exposure to solvent of the [4Fe-4S] cluster as a result of nucleotide binding, allowing chelator access. The kinetics of MgATP-induced Fe chelation from both wild-type and K15R FeP revealed two points. First, the K15R FeP does not show any Fe chelation prior to nucleotide addition, confirming that changing Lys-15 to Arg does not result in conformational changes that expose the [4Fe-4S] cluster to the chelator. Second, the addition of MgATP does not result in Fe chelation from K15R FeP, consistent with a lack of nucleotide-induced conformational changes.

MgATP and MgADP Binding to the K15R FeP

The lack of changes in the EPR line shape and the lack of a nucleotide stimulation of Fe chelation for the K15R FeP is similar to results obtained with the K15Q FeP, suggesting in both cases that MgATP binding does not result in conformational changes communicated to the [4Fe-4S] cluster. The lack of a conformational change in the K15Q FeP was not the result of a lack of MgATP binding since K15Q FeP was found to still bind MgATP, although with a somewhat reduced affinity compared to wild-type FeP (33% of wild-type) (18) . It was, therefore, necessary to examine the affinity of MgATP binding to the K15R FeP. Fig. 2(panel A) shows the ratio of MgATP bound per FeP at different MgATP concentrations determined by an equilibrium column binding method. Fitting the wild type FeP data to the Hill equation revealed an apparent dissociation constant (K ) of 571 µM. Under the same conditions, the K15R FeP showed no detectable binding of MgATP (Fig. 2, panel A). The sigmoidal nature of MgATP binding to the wild type FeP suggests cooperativity between the binding of nucleotides to the two binding sites, as has been previously suggested (15, 30, 31). The fit to the Hill equation resulted in a cooperativity factor of 2.1, supporting strong cooperativity in the binding of the two nucleotides. Given the strong cooperativity, the observed apparent dissociation constant (K ) represents the product of the two true dissociation constants.


Figure 2: MgADP and MgATP binding to wild type and K15R FePs. Equilibrium column binding of nucleotides to wild type FeP or K15R FeP were performed as described under ``Materials and Methods.'' Panel A, MgATP binding to wild type () or K15R () FeP. Panel B, MgADP binding to wild-type () or K15R () FeP. The ratio of the concentration of nucleotide bound divided by the FeP concentration was plotted against the concentration of free nucleotide. In each case, 4 mg of FeP was loaded onto the column and collected into one fraction. The data were fit to the Hill equation (F = M[S]/(K + [S])), where F is the fraction of nucleotides bound per FeP, M is the maximum number of nucleotides bound per FeP, [S] is the free nucleotide concentration, and n is the cooperatively factor. For wild type FeP, apparent dissociation constants (K) of 143 µM for MgADP and 571 µM for MgATP and values of n of 2.0 and 2.1 were determined.



We previously found that the affinity of the K15Q FeP for MgADP binding was nearly identical to that observed for wild-type FeP (18) . Fig. 2(panel B) shows the ratio of MgADP bound per FeP at different MgADP concentrations determined by an equilibrium column binding method. Fitting the wild type FeP data to the Hill equation revealed an apparent K of 143 µM. Again, the sigmoidal nature of the data suggested cooperativity in the binding of MgADP to the two binding sites, which was supported by a value of n = 2.0 for the fit to the Hill equation. In contrast to the binding of MgADP to the wild type FeP, the K15R FeP showed an extremely low affinity for MgADP even at concentrations as high as 300 µM. This extremely low affinity for MgADP contrasts with the normal affinity observed for the K15Q FeP (18) . Control binding experiments in which Mg was omitted revealed no detectable binding for either the wild type or K15R FePs.

Previous reports on the affinities of MgATP and MgADP binding to the reduced wild type FeP have suggested apparent dissociation constants ranging from 220 to 1710 µM for MgATP and 44 to 91 µM for MgADP (32) . The disparity in these reported values probably reflect limitations in the various methods that have been used to determine these values. We have found that particular care must be taken in the use of equilibrium dialysis methods. During the 6 or more hours needed to reach equilibrium, we have found significant hydrolysis of MgATP (up to 20%) to MgADP. The presence of MgADP in the binding assay would have a significant effect on the apparent binding constant for MgATP. In addition, because A. vinelandii FeP can self-oxidize dithionite (33) , the oxidized state of the FeP must be used. Advantages of the equilibrium column binding technique described in this work include the short time required for the attainment of equilibrium. This allows the maintenance of dithionite in the column buffer and thus the reduced state of the FeP. In addition, the short time reduces the amount of MgATP hydrolysis. The high performance liquid chromatography method used to detect the nucleotide concentrations provides a sensitive way to detect any hydrolysis of MgATP to MgADP or MgAMP. Using the column binding technique, nucleotide hydrolysis was determined to be less than 4%.

MgADP-induced Conformational Changes in the K15R FeP

Binding of MgADP to the FeP has been shown to induce changes in its [4Fe-4S] cluster (12, 34) . These changes are clearly different from those induced by MgATP. For example, addition of MgADP does not result in EPR line shape changes or in chelation of the iron from the cluster. MgADP binding does result in changes in the circular dichroism (CD) spectrum of the oxidized FeP which is different from changes induced by MgATP binding. Likewise, MgADP binding results in a change in the midpoint reduction potential of the [4Fe-4S] cluster from -310 to -430 mV (12). We examined the K15R FeP by both CD spectroscopy and microcoulometry as additional ways to probe both the integrity of the [4Fe-4S] cluster and as more sensitive ways to detect MgADP-induced conformational changes.

Fig. 3 (panel A) shows reduction curves for wild type FeP in the absence and presence of either MgATP or MgADP. From the fits of the data to the Nernst equation, midpoint reduction potentials of -290, -420, and -430 mV versus the standard hydrogen electrode were determined, respectively. In all cases, the maximum number of electrons per protein were found to be between 0.9 and 1.0, consistent with a single electron reduction of the [4Fe-4S] cluster from the 2 to the 1 state. The results for reduction of the K15R FeP are shown in Fig. 3, panel B. In the absence of nucleotides, the midpoint reduction potential for the K15R FeP was found to be -290 mV, similar to the wild type FeP. This data confirms that changing Lys-15 to Arg does not result in detectable changes in the [4Fe-4S] cluster. In contrast to the results for wild type FeP, the inclusion of MgATP or MgADP to K15R FeP was found to have no effect on the midpoint potential of the K15R FeP (Fig. 3, panel B).


Figure 3: Reduction curves for wild type FeP and K15R FeP with or without nucleotides. Microcoulometric electrochemical reduction of oxidized wild type or K15R FeP was performed as described under ``Materials and Methods.'' Panel A, reduction curves for wild-type FeP in the absence of nucleotides (), in the presence of 3 mM MgATP () or 3 mM MgADP (). Midpoint potentials were determined to be -290, -420, and -430 mV, respectively. Panel B, reduction curves for K15R FeP in the absence of nucleotides (), in the presence of MgATP (), or in the presence of MgADP (). All reduction midpoint potentials for K15R FeP were found to be -290 mV. Potentials are reported relative to the standard hydrogen electrode (SHE). Fits of the data to the Nernst equation are shown. In all cases, the maximum number of electrons was between 0.9 and 1.0 electrons per FeP.



Previous studies have shown that the circular dichroism (CD) spectrum of FeP in the visible wavelength region is a sensitive measure of the environment around the [4Fe-4S] cluster (34) . The addition of MgATP or MgADP to the FeP results in significantly different CD spectra, indicating different conformational changes. Fig. 4 (panel A) shows the CD spectra for oxidized, wild-type FeP both with and without added MgADP or MgATP. As reported (34) , the binding of nucleotides results in significantly different CD spectra. A similar experiment with K15R FeP (Fig. 4, panel B) revealed that in the absence of added nucleotides, the oxidized K15R FeP CD spectrum was nearly identical to the oxidized wild-type FeP. These results support the conclusion that changing Lys-15 to Arg does not change the environment around the [4Fe-4S] cluster, even when the [4Fe-4S] cluster is in the oxidized state. As predicted, the addition of MgATP or MgADP to the K15R FeP did not result in significant changes in the CD spectrum. This supports the conclusion that MgATP and MgADP do not induce conformational changes within the K15R FeP. A small decrease in ellipticity was observed at about 360 nm for the K15R FeP in the presence of MgADP.


Figure 4: Circular dichroism spectra of wild type FeP and K15R FeP in the presence or absence of nucleotides. The visible circular dichroism (CD) spectra of oxidized wild type FeP or K15R FeP were recorded as described under ``Materials and Methods.'' Panel A, CD spectra of wild type FeP in the absence of nucleotides (WTox) or in the presence of MgADP or MgATP. Panel B, CD spectra of K15R FeP in the absence of nucleotides (K15Rox) or in the presence of MgADP or MgATP. All spectra were baseline subtracted.




DISCUSSION

MgATP binding and hydrolysis are an integral part of the substrate reduction mechanism of nitrogenase (4, 16) . The results presented in this work support a model in which Lys-15 of A. vinelandii FeP plays a central role in nucleotide interactions with nitrogenase, functioning in binding of both MgATP and MgADP and in the conformational changes induced by both nucleotides.

MgATP interactions with nitrogenase include MgATP binding to the FeP followed by MgATP hydrolysis by the FePMoFeP complex. The FeP alone is known to bind 2 MgATP, one per subunit. This binding to the FeP results in conformational changes which appear to make the FeP competent for interaction with the MoFeP. The MgATP-induced conformational changes have been monitored by several methods, most of which detect changes in the [4Fe-4S] cluster (12, 29, 34, 35, 36, 37) . From the x-ray crystal structure and previous site-directed mutagenesis studies on FeP, it is known that the nucleotide-induced conformational changes within the FeP must be communicated over a distance of at least 19 Å from the nucleotide binding site to the [4Fe-4S] cluster (10, 18) . These changes have been suggested to be a prerequisite to the correct docking of the FeP to the MoFeP prior to MgATP hydrolysis and intercomponent electron transfer (16, 17) . Once the FePMoFeP complex is formed, the hydrolysis of MgATP is somehow linked to the transfer of the electron from the FeP to the MoFeP and possibly directly to substrate reduction (16, 18) . The exact mechanisms for how MgATP activates the FeP for binding to the MoFeP and how MgATP hydrolysis is linked to electron transfer remains largely undefined. It is becoming clear, however, that conformational changes within the FeP, and probably within the MoFeP, play an integral part in this mechanism.

In our previous work, the function of Lys-15 was probed by analysis of an FeP in which this residue was changed to the neutral amino acid glutamine (K15Q). The K15Q FeP was found to bind MgADP with normal affinity, clearly suggesting that the side chain of Lys-15 did not participate in MgADP binding (18) . In contrast, MgATP binding was affected, decreasing to 35% of wild type. This suggested that Lys-15 functioned in interaction with the -phosphate of MgATP. The K15Q FeP did not undergo conformational changes as detected by EPR or ,`-dipyridyl chelation. This lead to the conclusion that Lys-15 played a central role in nucleotide-induced conformational changes. Analysis of the x-ray structure of the FeP suggests that the -amino group of Lys-15 forms a salt bridge with the carboxylate of Asp-125 (Fig. 5A). Asp-125 is part of a second nucleotide binding pattern often observed in nucleotide binding proteins (Walker B) (18, 27) . Changing the conserved Asp-125 to Glu (D125E) was found to have dramatic effects on nucleotide-induced conformational changes within the FeP, suggesting that Asp-125 could provide a linkage from the nucleotide binding site to the [4Fe-4S] cluster (27) . This linkage has been suggested to be mediated through the amino acids between Asp-125 and Cys-132, a ligand to the [4Fe-4S] cluster (27) .


Figure 5: Stereoscopic view of the possible interactions of FeP Lys or Arg at position 15 with Asp-125. Molecular models were generated using the program Molecular Images (US Science, San Diego, CA) from the x-ray coordinates of the A. vinelandii FeP (10). A, model of the possible interactions of Lys-15 with Asp-125 in the proposed phosphate binding site of the FeP. B, substitution of Lys-15 with Arg showing possible interactions of the Arg side chain with the carboxylate side chain of Asp-125.



The results presented in this work on the properties of an FeP in which Lys-15 has been changed to an Arg can be integrated into the current model of the phosphate binding site and the function of Lys-15 in this interaction. In the absence of a bound nucleotide, Lys-15 and Asp-125 would be expected to form a salt bridge, neutralizing the charged residues in the interior of the FeP and stabilizing conformational changes mediated through Asp-125 (Fig. 5A). Upon binding of MgATP to the FeP, the salt bridge between Lys-15 and Asp-125 would be expected to be broken, with Lys-15 now interacting with the negatively charged phosphate groups (- and -) and Asp-125 with the Mg bound to the - and -phosphates of MgATP. The movement of Asp-125 to accommodate the bound nucleotide could result in a conformational change in the [4Fe-4S] cluster by changes in the relative position of Cys-132 (27) . The K15Q FeP would not be expected to form this salt bridge to the Asp-125. To probe the potential importance of this salt bridge, it was of interest to change Lys-15 to the positively charged amino acid Arg. This change would maintain the positive charge, but would change the nature of the interaction. A significant finding of the present work is that changing Lys-15 to Arg in the FeP greatly reduces K15R FePs affinity for both MgATP or MgADP. In addition, changing Lys-15 to Arg was found to prevent nucleotide-induced conformational changes. These results can be integrated into the model outlined above. Arg-15 is expected to form a salt bridge with Asp-125, as did the wild type Lys-15 (Fig. 5B). The nature of the interactions of Arg-15 with Asp-125 and with nucleotides is, however, clearly different from Lys-15. One possibility is that Arg-15 forms a stronger salt bridge with Asp-125, preventing the opening of the phosphate site to accommodate the phosphate groups of the incoming nucleotide. Consistent with this model, the guanidino group of Arg is more basic than the -amino group of Lys and also has the potential to form a hydrogen bond with the carboxylate of Asp as well as a charge interaction. The net effect could be to ``lock'' the active site closed, preventing either MgATP or MgADP from binding. Another related explanation is that to maintain an optimal distance for the salt bridge between Asp and the longer Arg, the protein backbone must shift to accommodate the 1-Å increase in length. This movement could move other amino acids critical for nucleotide binding out of place, thus having multiple effects on binding energetics. It should be noted, however, that no conformational changes in the EPR, CD, or midpoint potential of the cluster could be detected for the K15R FeP, suggesting that any conformational changes resulting from the change of Lys-15 to Arg are either very small or confined to the environment around the phosphate site. The formation of this tighter interaction between Arg-15 and Asp-125 could stop the nucleotide-induced conformational change proposed to be mediated through Asp-125.

In conclusion, this study provides evidence for a central role of Lys-15 of A. vinelandii FeP in interaction with both MgADP and MgATP and suggests a possible mode of interaction between Lys-15 and Asp-125.


FOOTNOTES

*
This work was supported by National Science Foundation Grant MCB-9315835 (to L. C. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300. Tel.: 801-797-3964; Fax: 801-797-3390; Internet: Seefeldt@cc.usu.edu.


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