An EPR Investigation of the Products of the Reaction of Cytosolic and Mitochondrial Aconitases with Nitric Oxide*

(Received for publication, May 8, 1997, and in revised form, June 11, 1997)

M. Claire Kennedy Dagger , William E. Antholine § and Helmut Beinert

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, the § Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and the   Institute for Enzyme Research, Graduate School and Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53705

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Cellular studies have indicated that some Fe-S proteins, and the aconitases in particular, are targets for nitric oxide. Specifically, NO has been implicated in the intracellular process of the conversion of active cytosolic aconitase containing a [4Fe-4S] cluster, to its apo-form which functions as an iron-regulatory protein. We have undertaken the in vitro study of the reaction of NO with purified forms of both mitochondrial and cytosolic aconitases by following enzyme activity and by observing the formation of EPR signals not shown by the original reactants. Inactivation by either NO solutions or NO-producing NONOates under anaerobic conditions is seen for both enzyme isoforms. This inactivation, which occurs in the presence or absence of substrate, is accompanied by the appearance of the g = 2.02 signals of the [3Fe-4S] clusters and the g approx  2.04 signal of a protein-bound dinitrosyl-iron-dithiol complex in the d7 state. In addition, in the reaction of cytosolic aconitase, the transient formation of a thiyl radical, gpar  = 2.11 and gperp = 2.03, is observed. Disassembly of the [3Fe-4S] clusters of the inactive forms of the enzymes upon the anaerobic addition of NO is also accompanied by the formation of the g approx  2.04 species and in the case of mitochondrial aconitase, a transient signal at g approx  2.032 appeared. This signal is tentatively assigned to the d9 form of an iron-nitrosyl-histidyl complex of the mitochondrial protein. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g = 2.02 [3Fe-4S] proteins.


INTRODUCTION

The aconitases (EC 4.2.1.3) are a family of dehyratases that catalyze the reversible isomerization of citrate and isocitrate via cis-aconitate (1). These enzymes, of which bovine mitochondrial aconitase (m-acon),1 is the most extensively studied, contain unique [4Fe-4S] clusters in that one of the irons, Fea, is not ligated to a protein residue but rather to a hydroxide from solvent (2). This is the same iron to which substrate binds during turnover. Inactivation of these enzymes occurs when Fea is lost by oxidation of the Fe-S cluster with the formation of a cubane [3Fe-4S] cluster that is detectable by electron paramagnetic resonance spectroscopy (EPR) at very low temperatures at g approx  2.02. To date there is no EPR evidence that this reaction occurs in vivo.

The recent discovery that the apo-form of mammalian cytosolic aconitase (c-acon), is identical to an RNA-binding protein, iron-regulatory protein (IRP1), has led to increased interest in the study of this isoform of the enzyme (3-5). There is a second IRP, IRP2, with similarities both in structure and function to IRP1 that will not be dealt with in this study (6, 7). These iron-regulatory proteins bind to specific stem-loop structures called iron-responsive elements which occur in the untranslated regions of the mRNA of a number of proteins involved in iron and energy metabolism (8). When bound to iron-responsive elements located in the 5'-untranslated regions of the mRNA as occurs with the iron storage protein, ferritin, translation is blocked (9). In contrast to this, binding to the iron-responsive elements in the 3'-untranslated regions of the mRNA of the transferrin receptor stabilizes the message, allowing translation to occur (10). For example, in iron-deficient cells the binding of IRP1 (apo-c-acon) to these iron-responsive elements would increase the production of transferrin receptors, which are key for increasing intracellular iron concentrations, while decreasing the biosynthesis of ferritin. Of particular interest in this process of cellular iron regulation is the mechanism of the interconversion between c-acon, containing a [4Fe-4S] cluster, and the cluster-free IRP1, since it has been demonstrated that the de novo biosynthesis of protein is not involved (11). Furthermore, in vitro experiments have shown that the Fe-S cluster of c-acon is quite stable, particularly in the presence of substrate (3) which can be assumed to be present in all cells. Therefore, any mechanism proposed for the interconversion must take these facts into consideration.

Two lines of evidence have led investigators to examine whether or not NO plays a role in the cellular process where c-acon is converted to IRP. First, it had been known from the work of Hibbs and co-workers (12, 13) that, when tumor target cells are cocultivated with activated macrophages, there is a loss of iron from the target cells that is associated with the inhibition of mitochondrial respiration and DNA replication as well as with the inactivation of m-acon. Later it was shown that this process occurs through an arginine-dependent pathway (14, 15). This pathway is now widely recognized to result from the production of NO formed by the action of the inducible isoform of the enzyme, nitric oxide synthase (16). The second line of evidence implicating NO came from the results of studies performed prior to the discovery and awareness of the importance of NO as a biologically active molecule with all its manifold functions. NO had been used in a variety of research endeavors, particularly in the area of transition metal chemistry, in which the addition of NO, with its unpaired electron, often resulted in products that were amenable to study by EPR. Early on, Vanin and Commoner (17-19) and their co-workers had described the so-called "g approx  2.04" signal observed for complexes generated synthetically by reaction of NO with iron and cysteine or found in cells treated with exogenous NO. This signal is attributed to a dinitrosyl-iron-dithiol complex with thiol groups provided by either low molecular weight compounds or by protein. In addition, it had been demonstrated by others that this g approx  2.04 signal can be observed when NO is added to some Fe-S proteins (20, 21). In more recent cellular experiments, where cells have been induced to produce NO, it has been shown that the activities of some Fe- or Fe-S-containing proteins are often decreased and moreover that these cells, when examined by EPR, display the typical g approx  2.04 signal (22, 23). Thus, in 1993 there were two reports demonstrating that the induction of nitric oxide synthase in macrophages by interferon-gamma and lipopolysaccharide to produce NO was accompanied by a loss of aconitase enzyme activity with a concomitant rise in the level of IRP1 binding to RNA (24, 25). These initial findings, implicating NO as an active agent in the conversion of c-acon to IRP1, have been further supported by a number of other cellular investigations (26-29).

We report here results of our in vitro studies on the reaction of NO with purified (>= 95%) bovine m- and c-acon in both the 3Fe and 4Fe forms by following enzyme activity and by observing EPR detectable species formed. Contrary to the results reported in previous publications which stated that activity is not lost when NO is added to aconitase (30, 31), we demonstrate both loss of activity and evidence by EPR regarding the disassembly of the Fe-S cluster. As there is experimental evidence supporting the intracellular formation of peroxynitrite from NO and superoxide anion (32), we have also investigated by EPR the effect of peroxynitrite on the active forms of both enzymes.


EXPERIMENTAL PROCEDURES

Materials and General Methods

Bovine heart mitochondrial aconitase and bovine liver cytosolic aconitase were purified according to published procedures (3, 33). Protein purity (>= 95%) was established by SDS-polyacrylamide gel electrophoresis and the cluster content was determined from iron, sulfide, and protein analyses by described methods (34-36). Active enzyme was prepared immediately before use by anaerobically incubating protein, iron, dithiothreitol, and dithionite (33). To remove excess activating reagents, the protein solution was rapidly desalted anaerobically on G-50 Sephadex columns equilibrated with the appropriate buffer (0.1 M HEPES, pH 7.5, or 0.1 M MES, pH 6.6) (37). Enzyme activity was determined at 25 °C following the isocitrate to cis-aconitate reaction (33). Nitric oxide gas was obtained from Aldrich Chemical Co. and was passed through 10% KOH prior to use. Saturated NO solutions were prepared by bubbling NO gas through water or buffer solutions made anaerobic either by equilibration in the anaerobic chamber or by repeated evacuation followed by the addition of either argon or nitrogen gas. The NO concentration of these solutions (~1.8 mM) was determined using an NO selective electrode (World Precision Instruments Inc., Sarasota FL). NONOates were obtained from Cayman Chemical Co., Ann Arbor, MI. SpermineNONOate (sperNO) solutions were prepared anaerobically in 0.1 M TAPS buffer, pH 8.9, and stored at 77 K. Concentrations were verified spectrophotometrically (252 nm, epsilon = 8, 500 M-1 cm-1) before use (38). The rate of decay and concomitant NO release of sperNO was determined by following the decrease in absorbance at 252 nm using the specific conditions of the reaction being studied, i.e. buffer, pH, and additives. Peroxynitrite was prepared as described previously (39) and stored at 77 K. Prior to use, the solution was diluted with 10 mM NaOH to the desired concentration which was determined spectrophotometrically (302 nm, epsilon  = 1,670 M-1 cm-1). Care was taken that upon addition of the alkaline peroxynitrite solution the pH of the sample was not altered significantly. All other chemicals were reagent grade and were obtained from Aldrich Chemical Co.

EPR Spectroscopy

EPR spectra were recorded with an X-band Varian E112 century series spectrometer at room temperature, 77 K, and 10-20 K using standard methods. A frequency counter (EIP model 548) was used to determine the microwave frequency and the field positions of prominent features in the spectra were determined using a Gauss meter (MH-110R Radiopan, NMR Magnetometer) or by comparison of the magnetic field to positions so determined. Quantitation of spin concentrations were made using a 1 mM copper perchlorate standard by comparing the double integral of the spectra to that of the standard run under similar conditions. All EPR samples run at 77 K or 10-20 K were contained in calibrated quartz EPR tubes. Data manipulation of the final EPR spectra was performed using the graphing and data analysis program SUMSPC92, available from the National Biomedical ESR Center, Milwaukee, WI. To separate the signals of a spectrum (A), the following strategy was employed in obtaining the difference spectra: fractions of spectrum B of a single species are subtracted from spectrum A until negative peaks of the signal for B vanish.

Addition of NO to Aconitase

All manipulations were performed inside a Coy Anaerobic Chamber equipped with a gas analyzer sensitive to >= 2 ppm oxygen using anaerobic reagents, unless otherwise noted. Initial experiments were conducted using NO solutions which were added directly to the enzyme samples in 0.1 M HEPES/K+, pH 7.5. In some samples the order of additions was reversed. When sperNO solutions were used, the enzymes were in 0.1 M MES, pH 6.6, as preliminary experiments had established that the rate of NO release at this pH and at room temperature (t1/2 in the absence of enzyme = 62 min) was suitable for both EPR and activity measurements. It was also determined that the rate of NO release from sperNO at pH >=  8.5 and at 4-6 °C is negligible. For the experiments using sperNO, two different methods were employed. In method 1, following the addition of sperNO to the enzyme, EPR samples were taken at different time points, quenched by rapidly raising the pH to >= 8.5 upon addition into tubes which contained the residue of 20 µl of 2.0 M TAPS, pH 8.8, evaporated to dryness. The tubes were placed in an ice bath until frozen in liquid nitrogen. Similarly, activity measurements were made on samples that were first diluted into substrate at pH 8.5 and at 4-6 °C. In method 2, after addition of sperNO to the enzyme, the solution was placed in an EPR flat cell and the reaction followed either by recording repeated scans or by observing the increase in the g approx  2.04 signal. The first time point for these EPR experiments occurs at ~5 min; the time it takes to fill the flat cell, remove it from the anaerobic chamber and position it in the spectrometer. At the end of the reaction, i.e. when there was no further discernible increase in the EPR signal, the sample was transferred anaerobically from the flat cell to an EPR tube for examination and quantitation at low temperatures. Samples were reduced using aliquots of a 50 mM solution of dithionite in 0.1 M TAPS, pH 8.9.


RESULTS

Principal EPR Signals

EPR studies were performed at room temperature, at 77 K (liquid nitrogen), and at 10-20 K for identification and detection of species formed from the reaction of NO and aconitase. Although the [4Fe-4S]2+ cluster found in native, active aconitases is in the S = 0, EPR silent state, the loss of Fea by oxidation leads to inactivation of the enzyme with formation of the [3Fe-4S]1+ cluster which is readily observed by EPR at temperatures below ~30 K, at g approx  2.02 (2). Fig. 1 shows the spectra for the two bovine isoforms, c-acon and m-acon, which are sufficiently different to allow for their identification. In contrast, the EPR signals for the d7 (g approx  2.04) and d9 forms of the dinitrosyl-iron-thiol complexes of the protein can be detected at all temperatures (Fig. 2). The line shape of the g approx  2.04 signal is invariant at low and high temperatures as it arises from a protein-bound species, whereas the signals for the low molecular weight complexes at room temperature exhibit multiline patterns (17, 40, 41). The isotropic hyperfine interactions observed for the low molecular weight complexes are characteristic for rapidly tumbling paramagnetic species.


Fig. 1. EPR spectra of 3Fe c-acon and m-acon. EPR spectra of c-acon (32 µM) and m-acon (25 µM) in 0.1 M Hepes, pH 7.5. Conditions of spectroscopy: microwave power and frequency, 0.1 milliwatt and 9.177 GHz; modulation amplitude and frequency, 0.5 millitesla and 100 kHz; time constant, 0.128 s; scanning time, 5 millitesla/min; temperature, 12 K.
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Fig. 2. EPR spectra of the d7 and d9 forms of DNI-acon. A, EPR spectrum of product formed upon the anaerobic incubation of 120 µM [4Fe-4S] active m-acon with 8.3 mM sperNO, pH 6.5, at 23 °C for 30 min (no enzyme activity remaining). B, EPR spectrum of A following the addition of excess dithionite. Conditions of spectroscopy as in Fig. 1 except for: microwave frequency, 9.229 GHz; scanning time, 20 millitesla/min; and temperature, 15 K.
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Addition of NO Solutions to Aconitase

Initial experiments of the reaction of NO with aconitase were carried out by adding varying amounts of a buffered saturated solution of NO to active enzyme, either aerobically or anaerobically. The results showed a rapid inactivation of enzyme followed by a slower inactivation over time. In these experiments substrate afforded some protection but did not prevent inactivation. For example, when m-acon was incubated anaerobically with an NO solution at an NO:enzyme ratio of 65:1, in the absence of substrate, 56% activity remained at 5 min and 25% at 60 min. The corresponding values in the presence of substrate were 96 and 76%. This trend was seen for both enzymes under aerobic as well as anaerobic conditions. The main difference between the two isoforms of the enzyme is that it takes a higher concentration of NO to bring about a corresponding inactivation of c-acon when compared with m-acon. When these samples were examined by EPR, the main species detected at 77 K was the g approx  2.04 signal, typical for the dinitrosyl-iron-thiol complex (DNI-acon) (Fig. 2A) while at 10-20 K, varying intensities of the signal for the 3Fe cluster were also seen (data not given). For c-acon, a third signal to be discussed below, appeared in some of the samples. Although we consistently observed inactivation of the enzyme using this method, we were unable to obtain reproducible, quantitative results. As an example, the extent of inactivation of enzyme would differ when enzyme was added to the NO solution as compared with the reverse order of addition. Furthermore, adding NO solutions in a single dose does not simulate cellular conditions where NO can be generated continuously. Therefore, in the remainder of the experiments to be described NONOates were used as the source of NO.

Continuous Assay following Addition of SpermineNONOate to Aconitase

The results of experiments using method 2 (see "Experimental Procedures") to follow the reaction of active [4Fe-4S] m-acon and NO are shown in Fig. 3. From these results it is clear that there is a loss of activity and that this loss occurs prior to the appearance of the DNI-acon signal. As an example, the t1/2 for the loss of enzyme activity in the presence of 10 mM citrate was 20 min, whereas the time for the half-maximal height of the g approx  2.04 signal to develop was 40 min. It should be pointed out that this signal results from iron in the d7 state in the DNI-acon complex and that the Fe in the complex may also exist in the d6 or d8 EPR-silent states. The quantitation of these species by EPR is not possible and to be able to do so will require other more discriminating techniques such as Mössbauer spectroscopy. The EPR spectra at 77 K and 12 K of the samples obtained at the end of the room temperature reaction were the same as at room temperature, i.e. only the signal for DNI-acon was seen. Quantitation of this signal for a number of similar experiments gave amounts that were ~40% of the original enzyme concentration, although in one experiment it was as high as 60%. Similarly, when excess dithionite was added to these samples in an attempt to convert all of the DNI-acon to the d9 state (Fig. 2, lower spectrum) and when quantitation of the EPR signal was performed, the concentrations again were only ~40% of the original enzyme solution. The presence of 1.0 mM substrate in these reactions (Fig. 3) was ineffective in protecting the enzyme in that both the rate of inactivation and the rate of formation of DNI-acon were essentially the same as in the absence of substrate. A comment should be made about the apparent anomalous results showing that both inactivation of the enzyme and formation of the complex were faster in the presence of 10 mM citrate than in its absence (Fig. 3, A and B). By comparing the rates of NO release from sperNO in buffer, and buffer plus 10 mM citrate, this effect was shown to be due to the faster rate of decay of sperNO and NO release in the latter. The increase in the rate of decay closely parallels the increased rate of inactivation when compared with the substrate-free reaction. The rate of the decomposition of NONOates is dependent on a number of factors, not just pH and temperature.


Fig. 3. Time course of inactivation of m-acon and formation of DNI-acon in the presence of sperNO. SperNO, final concentration 4.5 mM, was added anaerobically to a solution of 200 µM [4Fe-4S] m-acon in 0.1 M MES at pH 6.6 and at 23 °C. A, continuous monitoring of the EPR signal at g = 2.04 was made using a flat cell at a microwave power of 100 milliwatts and at a modulation amplitude and frequency of 0.5 millitesla and 100 kHz. B, activity measurements of the same sample as in A were performed simultaneously as described under "Experimental Procedures."
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Consecutive Sampling following Addition of SpermineNONOate to Aconitase

A drawback in performing the EPR experiments at room temperature is that during the course of the reaction we obviously cannot detect those EPR signals observable only at low temperatures. As described in method 1 ("Experimental Procedures") experiments were thus performed using the 4Fe enzymes where samples were taken at different time points for low temperature EPR measurements while concomitantly performing activity assays. At 77 K the DNI-acon signal was seen in all the samples while at 10-20 K, in addition to the g approx  2.04 signal, signals of varying intensity for the cubane [3Fe-4S] cluster were also observed for the early time points (data not given). Upon quantitation, neither signal correlated with the loss of activity and, furthermore, the concentration of the 3Fe signal was much lower than could be accounted for from activity measurements. It appears that although varying amounts of the cubane 3Fe protein are formed during the reaction of both m- and c-acon, that it is not an obligatory intermediate. In support of this observation, some of the samples taken during the course of the reaction with residual activity, give signals only for DNI-acon. Since other samples contain both signals, it can be concluded that the reason for the absence of the 3Fe signal is not due to the rapid disassembly by NO of cubane 3Fe clusters once formed. This conclusion will be supported by results obtained from experiments of NO and 3Fe m-acon to be discussed below.

Formation of a Thiyl Radical with Cytosolic Aconitase and NO

As stated above, when a NO solution was added to active 4Fe c-acon, a third signal was observed in some samples at both 77 and 10-20 K (Fig. 4A and uppermost spectrum). Since the g approx  2.02 signal of the 3Fe cluster is not seen at 77 K it was possible to obtain g values and the line shape of the third signal by subtraction of the g approx  2.04 signal from spectra obtained at this temperature. The resulting spectrum with gpar  = 2.11 and gperp  = 2.03 (Fig. 4, A and B) has been assigned to the transient formation of a thiyl radical by comparison to similar signals described by Nelson and Symons (42). In one experiment the intensity of the signal was as high as 65% of the signal intensity for DNI-acon. Although we have not conducted a thorough search for the exact conditions as to when this signal appears, as demonstrated in Fig. 4, it can appear early in the reaction.


Fig. 4. Thiyl radical formation. Uppermost EPR spectrum is of a sample of 58 µM c-acon incubated for 15 min with 0.54 mM NO at 23 °C and pH 7.5. Activity remaining is 85% of control. A, spectrum of the sample used to obtain the uppermost spectrum rerun at 77 K. B, spectrum of DNI-acon as in Fig. 2A. The difference spectrum, A-B (see " Experimental Procedures"), is the spectrum of the thiyl radical. Conditions of spectroscopy as described in the legend to Fig. 2 except that the microwave frequency was 9.229 GHz for the uppermost spectrum and 9.080 GHz for A and B, resulting in different magnetic fields for the same g value. The abscissa applies to all spectra.
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When a reaction of 4Fe c-acon and sperNO (Fig. 5) was run under the same conditions as described above for m-acon (Fig. 3) using method 2, a similar pattern for the formation of the DNI-acon signal was observed. Also, as for m-acon, the presence of substrate did not substantially alter the rate of reaction. The primary difference between m-acon and c-acon is that under these conditions, the extent and pattern of inactivation are different. For c-acon a plot of activity versus time indicated a biphasic reaction (data not given) and at the end of the reaction (Fig. 5) 34 and 41% of the activity remained in the absence and presence of substrate, respectively, whereas for m-acon under the same conditions, none remained.


Fig. 5. Time course of the formation of DNI-acon during the inactivation of 4Fe c-acon with sperNO. Active (4Fe-4S) c-acon incubated with sperNO in the presence and absence of substrate at pH 6.5 and 23 °C. The concentration of enzyme in the absence of substrate was 66 µM and in the presence of substrate 76 µM. The ratio of sperNO to enzyme was 30:1 and conditions of spectroscopy as described in the legend to Fig. 3.
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Formation of a Dinitrosyl-Iron-Histidyl Complex with Mitochondrial Aconitase and NO

When sperNO was added to 3Fe m-acon at room temperature and the spectrometer set to follow increases in the intensity of the first peak appearing in the spectrum, traces as shown in Fig. 6A were obtained. It had been assumed that the spectrometer had been set to record increases in the peak of the g approx  2.04 species since a rough calculation of the g value from the spectrometer settings indicated this. Furthermore, in trial experiments at room temperature, only the signal for DNI-acon had been observed. However, it became apparent from the pattern of the traces obtained from following the reactions by EPR, that what was being observed was either a variation in the formation of DNI-acon or the transient formation of another EPR active species. When the experiment was repeated and the peak height of the signals was determined from repeated 4-min scans, the plot shown in Fig. 6B was obtained. It became evident that what was occurring was the formation and disappearance of a new signal which overlapped the g approx  2.04 signal of DNI-acon. The original traces (Fig. 6A) had been obtained at a position corresponding to the peak of the new signal which is located on the right shoulder of the signal for DNI-acon. The new signal reaches a maximum at ~15 min and is not detectable at 30 min. Scans A and B in Fig. 7 (23 °C) are for the 15- and 75-min time points, respectively, for the experiment shown in Fig. 6B. The presence of a second signal is obvious (Fig. 7A at 23 °C) and by subtraction for the contribution to the spectrum of the g approx  2.04 DNI-acon species (Fig. 7B at 23 °C) two new signals (Fig. 7, A-B, at 23 °C) are observed: one that is similar in line shape to that of d9 DNIC-acon (Fig. 2B) but with very different g values, 2.032 and 2.004 versus 2.006 and 1.970, and a second minor species with g values of 2.05 and 2.01. 


Fig. 6. Transient formation of an EPR active species during the reaction of 3Fe m-acon and sperNO. A, continuous monitoring of the anaerobic reaction of 95 µM [3Fe-4S] m-acon with 2.6 mM sperNO in the presence and absence of citrate. All other conditions as in Fig. 3A. B, the reaction in the absence of substrate was repeated as in A except that relative peak heights were determined from sequential 4-min scans.
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Fig. 7. EPR spectra obtained at 23 °C, 77 K, and 16.6 K, during the reaction of 3Fe m-acon and sperNO. Upper left, spectra obtained at 23 °C. Spectrum A is the scan at 15 min and spectrum B is the scan at 75 min of the reaction described in Fig. 6. A-B is the difference spectrum of A and B. Lower left, spectra obtained at 77 K of samples prepared as those in Fig. 6 except that A was frozen in liquid nitrogen at 15 min and B at 75 min. A-B is the difference spectrum of A and B at 77 K. Conditions of spectroscopy as in Fig. 4A. Right, spectra obtained at 16.6 K. Spectra A and D are from the same samples as A and B at 77 K. Spectrum B is of [3Fe-4S] m-acon at 16.6 K as in Fig. 1B. C is the difference spectrum of A and B, and E is the difference spectrum of C and D, all at 16.6 K. Conditions of spectroscopy as in Fig. 2. The 40 gauss marker applies to all spectra.
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To identify these species and also to determine if other EPR active substances that are seen only at low temperatures were present, samples were prepared under conditions as in the room temperature experiment and then rapidly frozen in liquid nitrogen at 10, 15, and 75 min. The EPR spectrum at 77 K for the 15-min time point (Fig. 7A at 77 K) again shows this second signal in addition to that for g approx  2.04 DNI-acon. Subtraction of the latter (Fig. 7B at 77 K) yields a spectrum (Fig. 7, A-B, at 77 K) very similar to that found at room temperature (Fig. 7, A-B, at 23 °C) indicating that no other species were being detected. Finally, these same samples were also examined by EPR at 16.6 K. In Fig. 7A at 16.6 K, the signal for 3Fe m-acon at g approx  2.02 is now seen superimposed upon the other two signals. Subtraction of the spectrum for 3Fe m-acon (Fig. 7B at 16.6 K) from the 15-min time point (Fig. 7A at 16.6 K) gives a spectrum (Fig. 7C at 16.6 K) not unlike the 77 K spectrum (Fig. 7A at 77 K). A scan of the sample at 75 min which at higher temperatures showed only the signal for DNI-acon now has a broad signal on the high-field side (Fig. 7D at 16.6 K). We assign this to excess unreacted NO in the sample (43). Finally, if we subtract this spectrum (Fig. 7D at 16.6 K)) from the previous difference spectrum (Fig. 7C at 16.6 K), we again obtain a spectrum (Fig. 7E at 16.6 K) for the new species with g values as those found at room temperature and 77 K. Analysis of the results at the three different temperatures all lead to the conclusion that two new EPR-detectable species are formed in the reaction of 3Fe m-acon and NO. The appearance of these signals at room temperature led us to believe that they might be due to the formation of a different iron-nitrosyl complex. We were aware from earlier work by the Commoner group (19), that they had synthesized iron-nitrosyl complexes with amino acid residues other than thiols. More recently, Chasteen and co-workers (44) in their studies of the reaction of ferritin and NO had observed a signal at gx' = 2.055, gy' = 2.033, and gz' = 2.015 that they assigned to a histidyl-iron-nitrosyl complex (44). Boese et al. (41) have seen a similar signal with iron, bovine serum albumin, and NO, and have also attributed it to a histidyl complex.

We thus prepared the iron-nitrosyl-imidazole complex by incubating anaerobically Fe2+ and imidazole with diethylamineNONOate. Dithionite was added to one of the samples. The resulting spectra are shown in Fig. 8. Subtraction of the spectrum of the reduced species (Fig. 8B) from the original spectrum (Fig. 8A) which is a mixture of two signals, gives a spectrum (Fig. 8, A-B) that we assign to the d7 state of the imidazole complex. This spectrum resembles one of the signals seen in all the final difference spectra of Fig. 7, i.e. the signal with g values of 2.050 and 2.17. The spectrum that most closely resembles the new signals observed in the reaction of [3Fe-4S] m-acon and NO with g values of 2.032 and 2.004 is that of the d9 state of the imidazole complex obtained upon reduction with dithionite (Fig. 8B). We thus tentatively assign these transient signals to the d7 and d9 forms of a histidyl-iron-nitrosyl-acon complex. The addition of sperNO to 3Fe c-acon gave no evidence for the formation of any species other than g approx  2.04 DNI-acon (data not given).


Fig. 8. EPR spectra of the d7 and d9 forms of the dinitrosyl-iron-imidazole complex. Spectrum of a solution prepared anaerobically in 0.1 M HEPES, pH 7.5, containing 10 mM imidazole, 0.5 mM ferrous ammonium sulfate, and 5.0 mM diethylamineNONOate and incubated 30 min at room temperature. B, spectrum of sample as in A to which an excess of dithionite has been added. A-B is difference spectrum of A and B (see "Experimental Procedures"). Conditions of spectroscopy are as described in the legend to Fig. 4A except that microwave frequency is 9.090 GHz and microwave power is 2 milliwatts.
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Optical Spectra of Reactants and Products

A number of experiments were performed to record changes in the optical spectra that occur during the reaction of sperNO and the various forms of aconitase. In Fig. 9 are shown the optical spectra for 3Fe and 4Fe m-acon and the product of the reaction of sperNO and 4Fe m-acon in which no activity remained. The inactivated enzyme sample, when examined by EPR, gave evidence for only the g approx  2.04 species. The optical spectrum for the product of the reaction of 3Fe m-acon and sperNO had features identical to that recorded for the 4Fe m-acon reaction. When the reactions of either 3Fe or 4Fe m-acon were followed by repeating 1-min scans from 300 to 700 nm, no isosbestic points were observed. Maximal changes in the spectra between reactant and products occur at 365 nm (increase) for 4Fe m-acon and 470 nm (decrease) for 3Fe m-acon.


Fig. 9. Optical spectra of [3Fe-4S] and [4Fe-4S] m-acon and 4Fe m-acon inactivated by sperNO. The concentrations of 3Fe and 4Fe m-acon were 30 µM. The spectrum of 4Fe + NO was normalized to the absorbance at 280 nm of the 3Fe and 4Fe forms. All spectra were run against a buffer blank of 0.1 M Hepes, pH 7.5.
[View Larger Version of this Image (18K GIF file)]

Reaction of Peroxynitrite and Aconitase

Addition of peroxynitrite to the 4Fe forms of both c-acon and m-acon resulted in inactivation of the enzymes and the appearance of the 3Fe g approx  2.02 signal in the EPR spectra obtained at 10-20 K (Fig. 1). No other signals were detected. Interestingly, in contrast to the reaction of NO and aconitase, substrate afforded protection to the protein. The aerobic addition of peroxynitrite to both c-acon and m-acon, at a ratio of 133:1, resulted in a 99% loss of activity for m-acon and 61% for c-acon in less than 3 min in the absence of substrate. In the presence of substrate both enzymes were completely protected with no loss of activity. As observed with the reaction of NO with the enzymes, c-acon requires higher concentrations of peroxynitrite to attain a similar level of inactivation as seen with m-acon. For example, the anaerobic addition of peroxynitrite to m-acon in a ratio of 50:1 resulted in a 32% decrease in activity at 1.5 min in the absence of substrate. Under identical conditions, for a ratio of peroxynitrite to enzyme of 100:1, c-acon lost 48% of its activity. It thus appears that peroxynitrite functions as other oxidants, inactivating the enzyme by oxidation of the [4Fe-4S]2+ cluster with loss of Fea and formation of the [3Fe-4S]1+ cluster. However, whereas the oxidative reaction of ferricyanide and m-acon is stoichiometric (45), this is not so with peroxynitrite. Apparently the rate of decomposition of peroxynitrite to form nitrate, t{ifrax,1,2] ~ 1 s (46), is faster than the rate of oxidation of the cluster. Finally, a limited number of experiments on the reaction of Obardot 2 and m-acon were performed. Inactivation of the enzyme occurred in the absence or presence of either superoxide dismutase or catalase and as reported previously by Flint et al. (47), is accompanied by the formation of the cubane 3Fe cluster.


DISCUSSION

It is apparent from the preceding sections that the number of conditions which can affect the course of the reactions that we studied is so large, that we had to limit ourselves and chose the conditions described. Of particular importance in this study is protein purity, and we thus recommend caution in comparing data obtained here to previous reports (30, 31).

Dinitrosyl-Iron-Thiol-Aconitase

A common feature of the reactions of NO and the various forms of m-acon and c-acon, is the appearance of the g approx  2.04 EPR signal assigned to an iron-nitrosyl-thiol-acon complex. Similar signals have been observed for synthetic iron-nitrosyl-cysteine complexes as well as for cellular preparations which have been exposed to added NO or NO produced endogenously (17-19, 22, 24). In a recent publication, Vanin (48) has clearly shown that the g approx  2.04 signal arises from iron in the d7 state, in contradiction to a report which attributed these signals to iron with a d9 configuration (49). Our results agree with those of Vanin, as reduction with dithionite of aconitase samples exhibiting the g approx  2.04 signal yielded EPR spectra (Fig. 2B) similar to those obtained by him for the d9 state of the cysteine complex. Many of the EPR spectra of cellular samples exhibiting the g approx  2.04 signal also contain signals suggestive of the d9 species (15, 22, 23).

Inactivation of Aconitase and Disassembly of the Fe-S Cluster by NO

Despite previous claims to the contrary (30, 31), we have clearly shown that NO is capable of inactivating both c-acon and m-acon when added anaerobically or aerobically to the active [4Fe-4S] form of either enzyme. The mechanism of inactivation does not appear to involve the obligatory formation of the cubane 3Fe cluster as is observed when a variety of different oxidants are added to active enzyme. For instance, the active 4Fe form of either enzyme, determined by activity, can exist simultaneously with both the inactive 3Fe form and the DNI-acon complexes as shown by EPR. In addition, there was no correlation between loss of activity and appearance of the 3Fe g approx  2.02 signal. The latter was always much less than could be accounted for by loss of activity. More importantly, when NO is added to the 3Fe form of m-acon, new transient signals appear during the early phases of the reaction and prior to the formation of DNI-acon (Fig. 7) that are not seen in the reaction of 4Fe m-acon. Also, as shown in Fig. 2, the pattern of the appearance of the DNI-acon signal for 4Fe m-acon is quite different from that seen under similar conditions for 3Fe m-acon (Fig. 6). We have tentatively assigned the signals for these transient species seen in the 3Fe m-acon reaction to the d7 and d9 forms of an iron-nitrosyl-histidyl-acon complex, with the d9 form predominating. The active site of m-acon, as determined by x-ray crystallography, contains three histidine residues in close proximity to the Fe-S cluster (50). As iron is being released from the cluster by the action of NO, it could ligate to these histidines and react with NO with formation of the transient complex. As further disassembly of the cluster occurs, it is possible that two of the three cysteines that are ligands to the Fe-S cluster, form an iron-thiol-nitrosyl-acon complex. The question remains as to why on reaction with NO, 3Fe c-acon does not form a similar histidyl complex, since both aconitases contain the same active site amino acid residues (51). It should be noted that it is not certain whether this histidyl complex contains two histidyl ligands or is a mixed complex with ligands from both histidine and cysteine (41).

Another notable difference between the two aconitases is the appearance of a thiyl radical in the reaction of NO and 4Fe c-acon. Although we have not made a thorough study of its formation and do not see it in all the samples examined, we can say it forms early in the reaction. We have observed it in at least four samples where 75% or more of the original enzyme activity remains. As shown in Fig. 4, uppermost spectrum, at temperatures below 20 K the EPR spectrum contains a mixture of three signals, including signals for 3Fe c-acon (g approx  2.02), the thiyl radical and g approx  2.04 DNI-acon. At 77 K only the last two signals are seen. However, as with 4Fe m-acon, while the reaction progresses, only the g approx  2.04 signal is observed. This was true for the last time points of the reaction shown in Fig. 5 even though 30-40% of the original 4Fe concentration remained, as was indicated from activity assays. The pattern of inactivation of c-acon in the presence of sperNO did not follow a simple decay as observed for m-acon (Fig. 3B) but exhibited a biphasic reaction. Also, as when NO solutions were used, it takes a larger molar ratio of sperNO to enzyme to inactivate c-acon in comparison to m-acon.

Electronic Spectra

In Fig. 9 are shown the optical spectra of 3Fe and 4Fe m-acon and the spectrum of the product of the reaction of NO with either form. The EPR spectra for the NO samples revealed only the g approx 2.04 signal and concentration of the solution, using an ultrafiltration unit, demonstrated that the chromophore was bound to protein. Exposure to air, or evacuating for 30 min at <0.1 atmosphere, resulted in no change in the optical spectrum. We would like to point out the difference between the spectrum of the purified 4Fe active m-acon shown here (Fig. 9) and that reported previously in one of the earlier studies claiming that NO does not inactivate the enzyme. These authors attributed the bands at 535 and 575 nm seen in the optical spectrum of the aconitase preparation used by them, to the active form of the enzyme (31). These bands are absent in the spectrum of 4Fe m-acon (Fig. 9). Having analyzed aconitase preparations similar to those used by the authors, i.e. obtained from Sigma, we assign these bands to contaminating heme compounds. This was confirmed by EPR where signals for heme were observed as well as by the presence of a very strong band at ~408 nm in the optical spectrum that shifted to ~424 nm on reduction with dithionite, behavior typical of the Soret band of heme (data not given).

Inactivation of Aconitase by Peroxynitrite

Inactivation of both aconitases by peroxynitrite is accompanied by the appearance of the g approx  2.02 EPR signal for the 3Fe clusters (Fig. 1). The limited extent of inactivation by peroxynitrite was surprising. It has been shown, for example, that 4Fe m-acon can be inactivated by titration with ferricyanide producing the 3Fe cluster stoichiometric to the amount of ferricyanide added (45). With peroxynitrite, the molar ratio of added solution to enzyme inactivated ranged from 22:1 for m-acon to 85:1 for c-acon. Also, substrate was very effective in protecting the enzyme. How to relate these results to what may occur in vivo is difficult for many of the reasons stated above. It can at least be stated that the source of the g approx  2.04 signal observed in many of the cellular studies involving NO, is not due to a reaction of aconitase with peroxynitrite.

Significance

The finding that both aconitases are susceptible to inactivation by NO in the presence or absence of substrate and furthermore, that the rates of these reactions are very similar, supports the significance of the results obtained from cellular studies. Inactivation of the mitochondrial enzyme can have profound effects on cellular energy production. The results of this study cannot answer questions concerning the role of NO in iron metabolism. At this point we do not know whether the species arising on reaction of NO with c-acon, which we have detected by EPR, are active as IRP; our quantitative evaluations leave room for the formation of EPR undetectable species, such as intact apoprotein that are active as IRP (4). Further work will be required to establish the quantitative relationships between the products of NO and aconitase, whether or not they are EPR-detectable. The aim of the present work was to establish beyond doubt that NO does react with the Fe-S cluster of aconitase.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant GM51831 (to M. C. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-4015; Fax: 414-266-8515; E-mail: ckennedy{at}mcw.edu.
1   The abbreviations used are: m-acon, mitochondrial aconitase; EPR, electron paramagnetic resonance spectroscopy; c-acon, cytosolic aconitase; IRP, iron-regulatory protein; DNI-acon, dinitrosyl-iron-aconitase, g approx  2.04 species; MES, 4-morpholineethanesulfonic acid; sperNO, spermineNONOate; TAPS, 3-[tris(hydroxymethyl)methyl]amino-1-propanesulfonic acid.

ACKNOWLEDGEMENTS

We acknowledge the use of the facilities of the National Biomedical ESR Center at the Medical College of Wisconsin where all of the EPR spectra were recorded (supported by National Institutes of Health Grant RR01008).


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