Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU.

The ATPase activity of HscA, a specialized hsp70 molecular chaperone from Escherichia coli, is regulated by the iron-sulfur cluster assembly protein IscU and the J-type co-chaperone HscB. IscU behaves as a substrate for HscA, and HscB enhances the binding of IscU to HscA. To better understand the mechanism by which HscB and IscU regulate HscA, we examined binding of HscB to the different conformational states of HscA and the effects of HscB and IscU on the kinetics of the individual steps of the HscA ATPase reaction cycle. Affinity sensor studies revealed that whereas IscU binds both ADP (R-state) and ATP (T-state) HscA complexes, HscB interacts only with an ATP-bound state. Studies of ATPase activity under single-turnover and rapid mixing conditions showed that both IscU and HscB interact with the low peptide affinity T-state of HscA (HscA++.ATP) and that both modestly accelerate (3-10-fold) the rate-determining steps in the HscA reaction cycle, k(hyd) and k(T-->R). When present together, IscU and HscB synergistically stimulate both k(hyd) (approximately = 500-fold) and k(T-->R) (approximately = 60-fold), leading to enhanced formation of the HscA.ADP-IscU complex (substrate capture). Following ADP/ATP exchange, IscU also stimulates k(R-->T) (approximately = 50-fold) and thereby accelerates the rate at which the low peptide affinity HscA++.ATP T-state is regenerated. Because HscA nucleotide exchange is fast, the overall rate of the chaperone cycle in vivo will be determined by the availability of the IscU-HscB substrate-co-chaperone complex.

HscA (heat shock cognate 66-kDa; Hsc66) is a specialized member of the hsp70 family of molecular chaperones that functions in the biosynthesis of iron-sulfur proteins. The biochemical properties of HscA, including slow intrinsic ATPase activity and nucleotide-dependent peptide binding (1)(2)(3), are similar to those of other hsp70s, but HscA displays distinct substrate and co-chaperone specificity. HscA interacts selectively with IscU, a 14-kDa protein proposed to serve as a scaffold for de novo assembly of Fe-S clusters (4,5), and may function to regulate Fe-S cluster formation and/or transfer to acceptor proteins. HscA recognizes a specific LPPVK sequence motif at positions 99 -103 of IscU and is able to bind synthetic peptides containing this sequence (6 -8). The crystal structure of the HscA substrate binding domain complexed with the peptide ELPPVKIHC was recently determined (9) and revealed that the peptide binds with the opposite orientation of that observed for DnaK peptide complexes (10). The interaction of IscU with HscA is regulated by the specialized J-type co-chaperone HscB (heat shock cognate 20-kDa; Hsc20), which escorts IscU substrate to the chaperone and enhances its binding affinity (4). The molecular mechanism whereby HscB enhances HscA and IscU binding is not known but appears to involve coupling of ATP binding and hydrolysis with conformational changes that regulate substrate affinity. ATP binding to HscA leads to formation of a tense state (T) with reduced substrate affinity, and subsequent hydrolysis to ADP and phosphate results in a relaxed state (R) with increased substrate affinity (2,3).
In an earlier study we characterized the kinetics of the individual steps of the HscA ATPase reaction cycle in the absence of auxiliary proteins to determine the intrinsic rates of conversion of HscA between its different substrate affinity states (Scheme 1) (11). HscA was found to bind ATP in a two-step process preceding ATP hydrolysis. The first step was binding of HscA and ATP, and the second was interpreted as a conformational change involving conversion of HscA from the R-to the T-state (HscA⅐ATP 3 HscA ‡ ⅐ATP). ATP hydrolysis occurs in the T-state, and this is followed by a conformational change that returns HscA to the R-state. ATP hydrolysis and the subsequent conformational relaxation are rate-limiting in the overall cycle and are Ͼ10 3 -fold slower than release of the products ADP and phosphate. Thus, in contrast to other hsp70 isoforms that are regulated at both ATP hydrolysis and ADP/ATP exchange (12), regulation of the HscA reaction cycle is expected to occur solely at the rate-determining hydrolysis step.
The interconversion of HscA between its different conformational states is regulated by HscB and IscU, but the effects HscB and IscU have on different kinetic steps of the HscA ATPase cycle have not been investigated. Individually, HscB and IscU weakly stimulate HscA steady-state ATPase activity (maximal stimulation Ͻ10-fold), but together they synergistically stimulate HscA activity Ͼ400-fold (1,4). In addition, HscB reduces the concentration of IscU required for half-maximal stimulation of HscA steady-state activity ϳ20-fold (4). Regulation of HscA ATPase and IscU binding activity may occur in a manner similar to that suggested for DnaK. The ATPase activity of DnaK is regulated by the hsp40-type cochaperone DnaJ, and DnaJ is proposed to mediate DnaK substrate specificity by targeting peptides to the low affinity Tstate of DnaK and enhancing the rate of ATP hydrolysis (13)(14)(15). Individually, DnaJ and peptide substrates stimulate DnaK k hyd weakly at physiological levels (14 -18), but when present together DnaJ and peptide substrates can stimulate k hyd Ն10 3 -fold (15,18). This synergistic stimulation is thought to result in conversion of DnaK to a high affinity conformational state that stabilizes the DnaK-substrate complex (13)(14)(15)18). The effects of DnaJ on DnaK activity appear to be mediated primarily by the N-terminal J-domain of DnaJ (15, 17, 19 -21), although other regions contribute to the interactions (19,22,23). The J-domain present at the N terminus of HscB may function similarly in activating HscA. Apart from the J-domain, however, HscB and DnaJ differ considerably and may have different regulatory actions. The C-terminal region of HscB consists of a compact three-helix bundle (24), whereas DnaJ contains Gly/Phe-rich and Cys-rich zinc finger regions that have been implicated in both chaperone and regulatory activities (19,22,25,26). In addition, HscB does not exhibit intrinsic chaperone activity (2) of the type displayed by DnaJ (25,(27)(28)(29), and HscB appears to be more specific than DnaJ in targeting proteins to its hsp70 partner (4,6).
To better understand the regulation of HscA by HscB and IscU, we have used a combination of affinity sensor, steadystate ATPase, single-turnover ATPase, and pre-steady-state stopped-flow kinetic measurements to examine their effects on HscA catalytic activity and nucleotide interactions.

EXPERIMENTAL PROCEDURES
Materials-Escherichia coli DH5␣FЈIQ cells were from Invitrogen; bacterial growth media components were from Difco; and other reagents were from Sigma. Recombinant HscA, HscB, and IscU were expressed and purified as described previously (1,4). Some purified preparations of HscB were found to contain small amounts of IscU that affected HscA ATPase activity; HscB samples were therefore passed over a column containing immobilized antiserum to IscU to remove contaminating IscU protein prior to kinetic analyses.
Steady-state ATPase Assays-ATPase rates were determined at 23°C in HKM buffer (50 mM Hepes, pH 7.5, 150 mM KCl, and 10 mM MgCl 2 ) by measuring phosphate release using either a colorimetric assay as described by Lanzetta et al. (30)  of the ATP is hydrolyzed) and initiating the reaction by addition of HscB or IscU. Aliquots of the reaction mixture (5 l) were removed at various times after initiating reactions and quenched by adding 10 l of acetonitrile (Fisher). The sample was mixed thoroughly and centrifuged at 2000 ϫ g for 30 s, and 2 l was spotted on polyethylene-cellulose TLC sheets (Supelco). TLC sheets were dried and developed in 1 M formic acid containing 0.5 M LiCl. The fraction of ADP present was determined using a PhosphorImager (Bio-Rad). Data were corrected for the background level of [␣-32 P]ADP (typically ϳ2%) and an unknown impurity (typically ϳ2%) that migrates farther than ADP on TLC. For those experiments in which HscB and IscU were added subsequent to ATP, the data were further corrected for the hydrolysis that occurred prior to addition.
ATP-induced Spectral Changes-Difference absorbance spectra were recorded using a Cary 1 spectrophotometer (Varian). Reactions containing HKM buffer and indicated levels of HscA, HscB, and IscU were incubated at 23°C for 5 min prior to initiating reactions by adding ATP. For kinetic measurements, reactions were monitored at 280 or 288 nm using a cuvette with a 1-mm path length. No spectral changes were observed upon mixing HscB or IscU with ATP.
ATP Binding Kinetics-Rapid kinetic measurements were performed as before (11) using a stopped-flow UV-visible spectrophotometer (Hi-Tech, UK). Assays were carried out in HKM buffer containing 0 -500 M ATP and indicated levels of HscA, HscB, and IscU. Experiments were initiated by mixing equal volumes of protein and nucleotide solutions that had been incubated at 23°C for 5 min. Absorbance changes were monitored at 280 or 288 nm, and experiments were repeated at least six times at each ATP concentration. The concentrations of proteins and nucleotides shown in the figures are final concentrations in the optical cell after mixing.
Isothermal Titration Calorimetry (ITC)-A Microcal Omega titration calorimeter (Microcal, Inc.) was used to investigate the binding of HscA and ADP at 25°C using procedures described previously (4,11). Data were corrected for buffer addition.
Surface Plasmon Resonance Analysis-SPR 1 methods were carried out at 25°C with a Biacore 3000 instrument (Piscataway, NJ) using methods described previously (3,4). HscB or HscA was randomly crosslinked to the surface of the sensor chip using amine coupling as recommended by the manufacturer. Analyte was injected over the sensor chip in HKM buffer containing either 1 mM ADP, ATP, or ADP and P i . Data are reported as changes in maximal relative response (response units (RU)) versus concentration of analyte injected. Samples containing ATP were mixed immediately before injecting to prevent significant hydrolysis of ATP. The percentage of bound ligand was calculated using the Equation 1, % bound ϭ 100⅐(RU analyte /M r analyte )/(RU bound ligand /M r bound ligand ) (Eq. 1) where RU analyte is the maximum response upon injection of the analyte; RU bound ligand is the amount of immobilized protein; and M r analyte and M r bound ligand are the molecular masses of the respective proteins used in the experiment.
Error Analysis-Kinetic values are reported Ϯ1 S.D. resulting from the fit to the data, with propagation of error through any subsequent calculations. Rate constants requiring a fit of k obs (i.e. k a and k d for ATP) are reported as Ϯ1 S.D. from the secondary fit, using all available values of k obs . Error bars are only shown in figures if 1 S.D. is greater than the symbol used.

Effects of HscB and IscU on k hyd -To investigate whether
k hyd contributes to the overall reaction rate in the presence of substrate and co-chaperone, we measured IscU and HscB effects on HscA activity under single-turnover hydrolysis conditions by monitoring the production of [␣-32 P]ADP from [␣-32 P]ATP. Experiments were performed using limiting [␣-32 P]ATP and high concentrations of HscA to ensure that most of the nucleotide would be bound rapidly compared with the rate of hydrolysis. For these experiments, we used levels of HscB (150 M) and IscU (150 M) several fold higher than their apparent affinities as determined previously in steady-state ATPase assays (K m HscB ϭ 5-12 M and K m IscU ϭ 2/34 M in the presence/absence of HscB; cf. Refs. 1, 2, and 4). Fig. 1, A-C, shows the time course of ATP hydrolysis at 23°C in the absence and presence of HscB and IscU (filled symbols). Previous studies showed that ATP hydrolysis by HscA is essentially irreversible (11), and the data are fit to a first order rate equation The rates of hydrolysis (k hyd ) observed in the presence of HscB (0.020 s Ϫ1 ) and IscU (0.006 s Ϫ1 ) were 10-and 3-fold greater, respectively, than observed for HscA alone (k hyd ϭ 0.002 s Ϫ1 ).
The insets in Fig. 1, A-C, show that steady-state assays performed under identical conditions, i.e. using high levels of cochaperone and substrate, yielded similar rates suggesting ATP hydrolysis remains rate-limiting in the presence of either HscB or IscU. Studies by others have shown that the order of addition of ATP and co-chaperones can complicate measurement of k hyd . Russell et al. (14) found that a kinetic step prior to ATP hydrolysis (e.g. a first order ATP-induced conformational change) can be rate-limiting when single-turnover experiments are performed by adding ATP to a mixture of DnaK and DnaJ. It is thus possible that the rate we observed for single-turnover experiments in which ATP was added to a mixture of HscA and HscB or HscA and IscU may not represent the actual rate of ATP hydrolysis. For this reason additional experiments were carried out in which HscA was preincubated for 1 min with limiting ATP prior to addition of HscB or IscU in order to allow for formation of low peptide affinity T-state (HscA ‡ ⅐ATP). The open symbols in Fig. 1, B and C, show that HscB and IscU stimulate HscA single-turnover hydrolysis activity to a similar extent regardless of the order of addition of ATP. These findings suggest that the rates observed in the presence of HscB or IscU represent rates of ATP hydrolysis.
We also investigated the effect of the combination of HscB and IscU on ATPase kinetics. When saturating levels of both auxiliary proteins were used in single-turnover experiments similar to those shown in Fig. 1, A-C, we found that Ͼ95% of the ATP was hydrolyzed at the first time point that could be acquired after initiating the reaction (ϳ5 s; data not shown). For this reason we instead carried out steady-state ATPase measurements to assess the effect of addition of both substrate and co-chaperone. Fig. 1D shows that addition of a combination of IscU and HscB causes a dramatic stimulation of HscA ATPase activity. The actual rate of ATP hydrolysis, k hyd , must be equal to or greater than the steady-state rate observed, k cat ϭ 1.14 s Ϫ1 . Thus HscB and IscU together stimulate k hyd to a much greater level (Х500-fold) than the sum of their individual stimulations (13-fold), indicating that HscB and IscU have a synergistic effect on k hyd . Conversion of HscA from the R-to the T-state (k R3T ) must also be stimulated by HscB and/or IscU because this kinetic step is only 70-fold faster than the rate of ATP hydrolysis in the absence of auxiliary proteins (11). It is not known, however, whether k hyd remains rate-limiting in the presence of HscB and IscU because other kinetic steps may be subject to regulation.
HscB Binds HscA ‡ ⅐ATP and Stimulates k T3 R -Previous studies have shown that J-type auxiliary co-chaperones typically interact only with the ATP-bound T-state of their cognate hsp70 (27,32,33) and enhance formation of the ADP-bound R-state by stimulating the rate of ATP hydrolysis (13-15 ,18). To investigate whether HscB binds to a unique nucleotide state of HscA, we examined ATP and ADP effects on the interactions of HscA and HscB by using SPR. In initial experiments, HscA was cross-linked to the surface of a sensor chip and exposed to HscB in the presence of either ATP or ADP. Fig. 2A shows that HscB binds immobilized HscA in the presence of ATP, and based on the strength of the signal observed, we estimated that ϳ22% of the immobilized HscA binds HscB by assuming for- mation of a 1:1 complex. In the presence of ADP, a much weaker signal was observed (ϳ1% binding), suggesting that HscB interacts weakly if at all with the ADP complex of HscA. A similar lack of binding was observed in separate experiments carried out in the presence of 1 mM ADP and 1 mM phosphate or in the absence of nucleotide (data not shown). Because immobilization of HscA could inhibit the ability of the ADPbound form of HscA to interact with HscB, SPR binding experiments were also carried out using immobilized HscB and exposure to soluble HscA (Fig. 2B). Again, binding of HscB and HscA was only observed in the presence of ATP, and no significant interaction was observed in the presence of ADP. These results indicate that HscB only binds with high affinity to the low peptide affinity T-state (HscA ‡ ⅐ATP; cf. Scheme 1).
The finding that HscB binds to the HscA⅐ATP complex suggested that HscB could regulate the kinetics of ATP binding and/or the RNT conversion. To determine whether HscB affects these steps, we made use of an ATP-induced spectral change in HscA. ATP binding to HscA gives rise to a transient shift in the near-UV absorption spectrum of HscA that can be used to monitor the rates of ATP binding and the R3 T conversion, and decay of the absorbance change can be used to monitor ATP hydrolysis and subsequent T3 R conversion (11). To investigate whether HscB affects the binding kinetics of HscA and ATP, we compared the rate of formation of the ATP-induced difference absorbance spectrum in the absence and presence of HscB by using stopped-flow methods. Our earlier stopped-flow experiments showed that HscA binds ATP in a two-step process as evidenced by a rapid, biphasic ATPinduced change in HscA absorbance (11). The initial rapid phase of this spectral change was dependent on ATP concentration and was interpreted as arising from formation of the HscA⅐ATP collision complex; the subsequent slower phase was independent of ATP concentration and was interpreted to result from conversion of HscA from the R-to the T-state (k R3T ; cf. Scheme 1). Fig. 3A shows a representative plot of the rate of the absorbance change following addition of 75 M ATP to a solution containing HscA and HscB. Residuals for single and double exponential fits to the data (Equations 3 and 4) indicate that the formation of the ATP-induced spectral transition is biphasic in the presence of HscB as was observed for mixing ATP with HscA alone (11). Experiments were carried out over a range of ATP concentrations in both the presence and absence of HscB, and the rates observed for the fast (k 1 ) and slow (k 2 ) spectral transitions are plotted as a function of ATP concentration in Fig. 3, B and C, respectively. As with HscA alone, the values obtained for k 1 in the presence of HscB increased with ATP concentration in a linear fashion. The slope (k a ATP ϭ 3.2 Ϯ 0.6 ϫ 10 4 M Ϫ1 s Ϫ1 ) and y intercept (k d ATP ϭ 1.1 Ϯ 0.6 s Ϫ1 ) obtained in the presence of HscB were similar to those observed in experiments performed using HscA alone (k a ATP ϭ 2.8 Ϯ 0.1 ϫ 10 4 M Ϫ1 s Ϫ1 , k d ATP ϭ 1.3 Ϯ 0.2 s Ϫ1 ), indicating that HscB does not affect the rate of ATP binding. The values obtained for the slow phase were independent of ATP concentration, and similar average values were obtained in the absence or presence of HscB (k 2 Х 0.10 s Ϫ1 ). Because k 2 is thought to represent a conformational change of HscA from the R-to the T-state (k R3T ), this result indicates that HscB does not affect the rate of formation of the T-state. These findings are consistent with the SPR results described above which indicate that HscB only binds to the T-state of HscA.
We also monitored the decay of the ATP-induced absorbance change to investigate the effect of HscB on k hyd and the rate of conversion of HscA back to the R-state (k T3R ). Fig. 4A shows time-dependent difference absorbance spectra produced by addition of ATP to HscA in the presence of HscB. These spectra are similar in shape and magnitude to those observed for HscA and ATP in the absence of HscB (⌬⑀ 280 ϳ2 ϫ 10 3 M Ϫ1 cm Ϫ1 (11)), indicating that the residue(s) giving rise to the underlying spectral shifts are insensitive to the binding of HscB. The difference absorbance spectra decay to a spectrum similar to that arising from mixing HscA and ADP, and we proposed that the decay observed reflects conversion of HscA from the low affinity T-state (HscA ‡ ⅐ATP) to the high affinity R-state (HscA⅐ADP) (11). To determine the rate of decay under conditions where most of the ATP is bound rapidly compared with ATP hydrolysis, we repeated this experiment by using high concentrations of HscA, HscB, and ATP. The sample absorbance under these conditions required use of a short path sample cell (1 mm) and monitoring reaction progress at 288 nm. As shown in Fig. 4B the rate of disappearance of the ATP-induced spectral change under these conditions can be described by a single exponential with k obs ϭ 0.0164 Ϯ 0.0003 s Ϫ1 . This rate is 7.8-fold faster than that observed in the absence of HscB (0.0021 Ϯ 0.0002 s Ϫ1 ; data not shown), indicating that HscB stimulates k T3R .
IscU Enhances R3 T and T3 R Conversion-We also made use of ATP-induced spectral changes to characterize the effect of IscU on the kinetics of HscA and ATP binding and the RN T interconversion. As found for HscB, mixing ATP and HscA in the presence of IscU also results in a time-dependent difference absorbance spectrum immediately following ATP addition (Fig.  4C). The data can be described by a single exponential, and the rate of decay (0.0063 Ϯ 0.0001 s Ϫ1 ) is 3-fold faster than that observed in the absence of IscU (0.0021 s Ϫ1 ). Thus IscU, like HscB, stimulates the rate of conversion of HscA ‡ ⅐ATP to the high affinity HscA⅐ADP R-state.
To investigate whether IscU affects the kinetics of ATP binding and/or the R3 T conversion, we examined the rate of formation of the ATP-induced difference absorbance spectrum using stopped-flow methods. Fig. 5A shows a representative plot of the rate of ⌬A 288 formation upon mixing ATP with a solution containing HscA and IscU. The data fit a single exponential with k obs ϭ 5.0 s Ϫ1 , and the residuals for this model (cf. inset, Fig. 5A) indicate the spectral change is monophasic under these conditions. This finding can be contrasted to the biphasic change observed for HscA alone (11) or in the presence of HscB (Fig. 3). To determine whether this spectral change arises from ATP binding or a subsequent unimolecular conformational change, we examined the rate of formation over a range of ATP concentrations. Fig. 5B shows that k obs is not affected by ATP concentration, indicating that it represents a unimolecular conformational change leading to formation of HscA T-state (k R3T ). The rate of this transition (5.0 s Ϫ1 ) is ϳ50-fold faster than that observed for HscA alone or HscA in the presence of HscB (Х0.1 s Ϫ1 ; Fig. 4). This indicates that binding of IscU strongly favors conversion of the R-state complex (HscA⅐ATP) to the T-state complex (HscA ‡ ⅐ATP).
Combined Effects of HscB and IscU on RNT Conversion-To characterize HscB and IscU effects on HscA conformational changes, we examined their combined effects on the ATP-induced spectral changes. In initial experiments ATP was added to a mixture containing HscA, HscB, and IscU, and spectral changes were monitored at 280 nm for ϳ10 s after mixing as described in Fig. 4. No difference absorption spectrum was observed, suggesting that if an HscA⅐ATP intermediate was formed in the presence of HscB and IscU it was short lived as expected for k cat Ͼ1 s Ϫ1 . To investigate this possibility, we monitored spectral changes at 280 nm in a stopped-flow spectrophotometer. Fig. 6A shows the results of rapid mixing of ATP with a mixture of HscA, HscB, and IscU. The data are fit to a double exponential model, and the residuals for this model (Fig. 6A, inset) indicate that the changes observed are biphasic. A transient increase in absorbance (k 1 ϭ 2.1 Ϯ 0.2 s Ϫ1 ) occurs following ATP addition as was observed in the presence of either HscB or IscU alone. However, the spectral transition rapidly decays with a rate (k 2 ϭ 0.13 Ϯ 0.01 s Ϫ1 ) that is ϳ60-fold faster than that observed with HscA alone (0.0021 s Ϫ1 ). Steady-state ATPase assays performed using identical protein concentrations yielded a rate (k cat ϭ 0.110 Ϯ.002) similar to k 2 , indicating that T3 R conversion and ATP hydrolysis occur concurrently in the presence of co-chaperone and substrate. The 60-fold stimulation of T3 R conversion by HscB and IscU together is greater than the individual effects of HscB (ϳ8-fold) or IscU (ϳ3-fold) (cf. Fig. 4, B and C). To investigate whether the initial absorbance increase results from ATP binding, we examined the dependence of k 1 on ATP concentration. Fig. 6B shows that the values obtained increase with ATP concentration in a linear fashion, indicating that the initial absorbance increase is associated with the bimolecular binding of HscA and ATP. The slope (k a ATP ϭ 3.8 Ϯ 0.6 ϫ 10 4 M Ϫ1 s Ϫ1 ) and y intercept (k d ATP ϭ 1.5 Ϯ 0.6 s Ϫ1 ) obtained in the presence of HscB and IscU are similar to those observed in experiments performed with HscA alone (k a ATP ϭ 2.8 Ϯ 0.1 ϫ 10 4 M Ϫ1 s Ϫ1 and k d ATP ϭ 1.3 Ϯ 0.2 s Ϫ1 ; cf. Table I). Thus, HscB and IscU together do not significantly affect the rates of ATP binding or release. The finding that HscB and IscU do not significantly alter the rates of ATP association and dissociation indicate that the decay rate (k 2 ) observed in Fig.  6A represents a lower bound on the rate of T3 R conversion. The stopped-flow experiments performed in the presence of HscB and IscU were limited to using low concentrations of ATP, where ATP binding is expected to limit the rate observed for k 2 .
HscB Is Released Following ATP Hydrolysis-In the absence of substrate, HscB binds to the T-state HscA ‡ ⅐ATP complex but binds weakly if at all to the R-state HscA⅐ADP complex (see Fig.  2). It is possible, however, that IscU stabilizes HscB binding to the HscA⅐ADP complex. To determine whether HscB is released from the HscA ‡ ⅐ATP-HscB-IscU complex following ATP hydrolysis and T3 R conversion, we carried out SPR experiments using immobilized HscA in the presence of either ATP or ADP. Fig. 7 shows that even when IscU is present HscB binds only to the HscA⅐ATP complex. In the presence of ADP (Fig.  7B), in contrast, addition of HscB and IscU together does not yield a signal significantly stronger than that of IscU alone. As observed previously, the rates of IscU binding and release are significantly faster in the T-state HscA ‡ ⅐ATP complex than in the R-state HscA⅐ADP complex (3). The finding that HscB interacts weakly if at all with the HscA⅐ADP-IscU complex suggests that HscB will be rapidly released following ATP hydrolysis and T3 R conversion even in the presence of substrate and that HscB will not exert effects at subsequent steps in the reaction cycle.
ADP Binding Kinetics-In the absence of auxiliary proteins, the rate of ADP/ATP exchange is more than 6,000-fold faster than ATP hydrolysis (11). HscB does not bind to the HscA⅐ADP complex (Fig. 3) and is therefore not expected to affect the kinetics of ADP binding. However, IscU does bind to HscA⅐ADP and may regulate the kinetics of HscA and ADP binding. We were unable to measure ADP binding kinetics directly, and we therefore used isothermal titration calorimetry to investigate the effect of IscU on ADP binding. The affinity observed in the presence of IscU (K D ϭ 158 M) was reduced 2.3-fold compared with that observed with HscA alone (K D ϭ 68 M). This decrease in affinity could result from a slightly faster rate of ADP release or the combined effects of a slower release rate with a larger decrease in binding rate. The very fast rate of release of ADP observed in the absence of IscU (60 s Ϫ1 ) (11), however, suggests that even in the presence of IscU the overall ATPase reaction rate is unlikely to be limited by the rate of ADP release.

DISCUSSION
In an earlier report we described the kinetics of the individual steps of the HscA ATPase reaction cycle in the absence of auxiliary proteins (11). In the studies described here, we have investigated the mechanisms whereby the co-chaperone HscB and the substrate protein IscU regulate the ATPase and substrate-binding activities of HscA. We have used several approaches including surface plasmon resonance and steadystate and pre-steady-state measurements. The kinetic constants determined for different steps shown in Scheme 1 in the presence and absence of HscB and IscU are summarized in  The steady-state ATPase rate is a lower bound on k hyd , since ATP hydrolysis is rate-limiting in the absence of auxiliary proteins. b ND, not determined. c The rate of T3 R conversion is a lower bound, since this kinetic step could only be measured using subsaturating levels of co-chaperone and substrate.   Fig. 8.
The HscA Chaperone Cycle-As is characteristic of hsp70type chaperones, HscA couples ATP binding and hydrolysis with conformational changes that control substrate affinity (2). ATP binding leads to formation of a tense state (T) with reduced substrate affinity and increased substrate exchange rates, and ATP hydrolysis to ADP and phosphate results in a relaxed state (R) with increased substrate affinity and decreased substrate exchange rates (3). In the absence of cochaperone and substrate, the steady-state reaction rate and cycling between R-and T-states is very slow (k cat ϭ 0.002 s Ϫ1 ). Under these conditions the rate of the cycle is determined by the rates of ATP hydrolysis (k hyd ) and T3 R conversion (k T3R ), and nucleotide exchange (k d ADP and k a ATP ) is Ͼ10 3 -fold faster than these steps (11). IscU enhances R3 T conversion (k R3T ), and IscU and HscB stimulate k hyd and k T3R such that the rate of cycling through the different conformational states can be accelerated more than 500-fold. Even in the presence of saturating amounts of substrate and co-chaperone, however, ATP hydrolysis and T3 R conversion remain rate-determining in the steadystate reaction cycle.
Substrate Binding-We showed previously that IscU is a specific substrate for HscA, binding to ADP and ATP complexes of HscA with K D values of 1.6 and 34 M, respectively (3,4). HscB also binds IscU and enhances HscA binding of IscU under steady-state-turnover conditions, lowering the concentration of IscU necessary for half-maximal stimulation of HscA ATPase activity to a level (K m ϭ 1.8 M) that is similar to the affinity of HscA⅐ADP for IscU. The SPR measurements presented here establish that HscB only interacts with the ATP complex of HscA suggesting that HscB acts specifically to recruit IscU to the low affinity T-state (HscA ‡ ⅐ATP). This type of role for HscB is supported by earlier findings with IscU mutants which showed that HscB could override binding defects caused by substitutions within the LPPVK recognition motif of IscU (7). Together, these results indicate that the ternary complex HscA ‡ ⅐ATP-HscB-IscU is more stable than binary complexes of HscA ‡ ⅐ATP with substrate or co-chaperone alone.
Substrate Capture-ATP hydrolysis measurements performed under single-turnover conditions revealed that HscB and IscU individually stimulate the rates of ATP hydrolysis and T3 R conversion only modestly, ϳ10and 3-fold, respectively. When both co-chaperone and substrate are present, however, k hyd and k T3R were synergistically accelerated to a level that is much faster than the additive effects of the individual proteins. In the presence of saturating levels of cochaperone and substrate, ATP hydrolysis occurred with a rate, ϳ1 s Ϫ1 , faster than IscU dissociation from immobilized HscA ‡ ⅐ATP (k obs ϳ 0.15-0.35 s Ϫ1 ; Fig. 7) (3). T3 R conversion is expected to occur concurrently with ATP hydrolysis, because k hyd and k T3R were observed to have essentially identical rates under all conditions assayed. These findings suggest that IscU will remain bound to HscA and be effectively trapped in the high affinity R-state HscA⅐ADP complex. HscB is expected to dissociate from this complex, because HscB does not interact with HscA⅐ADP even in the presence of IscU.
Substrate Release-IscU binds with high affinity to the Rstate HscA⅐ADP complex. This complex must dissociate ADP ϩ P i , bind ATP, and undergo R3 T conversion to regenerate the low affinity T-state HscA ‡ ⅐ATP. In the absence of IscU, the rate of this process is determined by R3 T conversion, because k R3T exhibits a rate that is 10 2 -fold slower than the intrinsic nucleotide exchange rate (ϳ10 s Ϫ1 ; cf. Ref. 11). The findings presented here show that IscU enhances R3 T conversion ϳ50fold. This effect reduces the lifetime of the HscA-IscU complex and serves as an auto-regulatory mechanism to allow additional cycles of substrate binding and release.
Comparison with the DnaK/DnaJ Chaperone System-The effects of IscU and HscB on HscA ATPase activity are in general similar to the effects of peptide substrates and DnaJ on DnaK. Peptide substrates stimulate DnaK ATP hydrolysis weakly (15)(16)(17)(18), and DnaJ, when used at concentrations approximating in vivo levels, exerts a weak stimulatory effect on DnaK ATP hydrolysis (14, 15 ,17, 18), Substrate-bound DnaJ dramatically accelerates the rate of DnaK ATP hydrolysis (15,18) as we find for IscU bound to HscB. These similarities suggest that DnaJ and HscB both tightly couple regulation of ATP hydrolysis to substrate capture by the high peptide affinity conformation of their respective chaperone partners. For DnaK, the large acceleration of ATP hydrolysis has been attributed to simultaneous interactions of the DnaJ J-domain with the DnaK ATPase domain and of the peptide substrate with the DnaK substrate binding domain (15,18,21,22). The similarity of the structure of the J-domain of HscB (24) to that of DnaJ (34,35) suggests that both co-chaperones regulate the ATPase activity of HscA by a similar mechanism. Despite differences in the orientation of the peptide region of IscU bound to substrate binding domain of HscA (9) compared with the peptide complex of DnaK (10, 36 -38), IscU appears to regulate the ATPase activity of HscA by a mechanism similar to that of DnaK substrates.
Although the mechanism of regulation of the ATPase activity and substrate capture by HscA and DnaK are similar, the kinetics and regulation of substrate release are different. Both HscA⅐ADP and DnaK⅐ADP substrate complexes require ADP/ ATP exchange and subsequent R3 T conversion to generate the low peptide affinity forms. For DnaK, nucleotide exchange is believed to determine the rate at which the low peptide affinity T-state (DnaK ‡ ⅐ATP) is regenerated, because ADP dissociation occurs with a rate that is significantly slower than R3 T conversion (16, 39 -41). In the case of HscA, nucleotide exchange and R3 T conversion are both fast (Ͼ1 s Ϫ1 ) and are orders of magnitude faster than DnaK ADP/ATP exchange. Thus, in the absence of auxiliary proteins, the half-life of a  Table I. See text for "Discussion." DnaK-substrate complex is expected to be much longer than that of the HscA-IscU complex. However, nucleotide exchange in DnaK is subject to regulation by GrpE, which accelerates ADP/ATP exchange and decreases the lifetime of bound substrates (42)(43)(44). In contrast, nucleotide exchange by HscA is not rate-limiting even in the presence of IscU and HscB and does not appear to be subject to regulation.
Comparison with Jac1 and Isu1 Regulation of Ssq1-Homologs of HscA (Ssq1), HscB (Jac1), and IscU (Isu1/Isu2) are present in yeast mitochondria (45)(46)(47)(48)(49) and like their bacterial counterparts (50 -53) have been implicated in the biogenesis of iron-sulfur proteins. Recent studies (50,51) have shown that Ssq1, Jac1, and Isu1 interact in a manner similar to that found for HscA, HscB, and IscU, suggesting that the major function(s) of this chaperone system have been conserved through evolution. Isu1 and Jac1 cooperatively stimulate Ssq1 ATPase activity, and Jac1 enhances the binding of Isu1 to ATP-bound Ssq1 (54). In addition, the evolutionarily conserved LPPVK motif in IscU that is recognized by HscA (6,7,9) is critical for Isu binding to Ssq1 (55). Phylogenetic analyses, however, suggest that Ssq1 is more closely related to DnaK than to HscA (56). Consistent with this, Ssq1 exhibits some ATPase reaction cycle kinetics that are more similar to those of DnaK than to HscA. Ssq1 forms nucleotide complexes that have greater stability than those of HscA, and Ssq1 nucleotide exchange is enhanced by the mitochondrial GrpE homolog Mge1. It is thus possible that eukaryotic mitochondrial chaperone systems involved in iron-sulfur protein biogenesis may employ somewhat different mechanisms of regulation.