H2O2-induced Kinetic and Chemical Modifications of Smooth Muscle Myosin

The effect of H2O2 on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of Pi release were measured by single-turnover kinetics. H2O2 treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, ∼50% of the molecules lost the ability to bind to ATP. H2O2 treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H2O2-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H2O2-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mm ATP, H2O2 caused a ∼50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H2O2 directly affects the force generating complex. Dithiothreitol treatment reversed the H2O2 inhibition of the maximal force by ∼50%. These data, when compared with the in vitro kinetic data, are consistent with a H2O2-induced loss of functional myosin heads in the muscle.

Contraction of smooth muscle is controlled largely by phosphorylation of the 20-kDa myosin regulatory light chain (RLC), 2 resulting in the cyclic attachment and detachment of myosin to actin (cross-bridge cycling) and the hydrolysis of ATP by actin-activated myosin ATPase (1). The level of RLC phosphorylation depends on the balance between the activities of MLCK and SMM phosphatase. MLCK is primarily activated (through Ca 2ϩ -calmodulin) by an increase in [Ca 2ϩ ] i from intracellular stores or influx of extra-cellular Ca 2ϩ , whereas inhibition of myosin phosphatase, which contributes to the amount of force at a given [Ca 2ϩ ] i (Ca 2ϩ sensitivity), occurs through a receptor-G protein-catalyzed signal transduction cascade (2)(3)(4)(5).
The mechanisms for H 2 O 2 inhibition of smooth muscle contraction are not well understood but have been proposed to involve effects on kinase-mediated signaling cascades, cytoskeletal effects, ionotropic effects, lipid oxidation, direct inhibitory effect on smooth muscle contractile proteins, and potentially confounding effects on epithelial cells (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In general, healthy smooth muscle is quite resistant to exogenous H 2 O 2 . The most extreme example is agonist activated bladder smooth muscle (bladder concentrations of H 2 O 2 often reach levels of 0.2-0.4 mM in healthy adults (21), which was shown to exhibit an EC 50 for relaxation of 36 mM H 2 O 2 (22)). Airway smooth muscle, although considerably more sensitive than bladder, still requires nearly mM concentrations of H 2 O 2 for relaxation of agonist induced contraction (23). Previously we showed that 0.1-3 mM H 2 O 2 -induced relaxation of ASM was caused by a reduction in the amount of force produced at given [Ca 2ϩ ] i , (Ca 2ϩ sensitivity), whereas [Ca 2ϩ ] i actually increased in response to H 2 O 2 treatment (23). Additionally, we showed that the inhibitory effect of peroxide on ASM contraction persisted after Triton X-100 permeabilization and RLC thiophosphorylation. Because the levels of RLC thiophosphorylation were unaf-fected by H 2 O 2 treatment, we proposed that H 2 O 2 directly inhibits the acto-myosin contractile apparatus (24).
Here we address whether or not myosin oxidation underlies the physiological effect of H 2 O 2 treatment in ASM as mentioned above. This is an important question because in terms of contractility it may not matter what the effects of H 2 O 2 are on signaling cascades upstream of myosin activation, because even pro-contractile-signaling would be nullified by direct inhibition of acto-myosin cycling. A better understanding of effects of peroxide on SMM will ultimately clarify the potential mechanisms at play in previous studies of H 2 O 2 -induced pathogenesis.
Here we characterize the effects of H 2 O 2 on the kinetics of purified SMM and correlate those data to changes in force generation in ASM tissue. We have examined the steady-state and single-turnover ATPase kinetics of purified SMM in both the unphosphorylated and phosphorylated states. We have used two soluble myosin subfragments that are suitable for kinetic experiments. HMM is double-headed and lacks two-thirds of the tail and therefore cannot form filaments. However, it is fully regulated by light chain phosphorylation. S1 is a single head domain that cannot form filaments and is not regulated by light chain phosphorylation. The effects of EDTA during the H 2 O 2 treatment and the effect of DTT after the H 2 O 2 treatment have been analyzed. In addition, we have analyzed the effect of H 2 O 2 on the total number of reactive thiols and the formation of intersubunit disulfide bonds. Mass spectral studies revealed which residues were oxidized and to which oxidation state. The effect of H 2 O 2 on the rate and amplitude of force development in permeabilized porcine tracheal smooth muscle strips was also examined. Remarkably, the specific effects on myosin kinetics observed for purified SMM were paralleled by the effects observed in the muscle, strongly suggesting that elevated H 2 O 2 levels during diseased states could alter myosin kinetics and partially explain altered contractility.

EXPERIMENTAL PROCEDURES
Protein Preparations-Protein preparations were essentially as described (25). HMM and S1 were prepared by digestion of gizzard SMM (26) with Staphylococcus aureus protease V8 (Sigma). Both HMM (E (0.1%) ϭ 0.65) and S1 (E (0.1%) ϭ 0.75) are mixtures of full-length heavy chains and clipped heavy chains with an internal cleavage at the actin binding loop ϳ26 kDa from the head-tail junction. The light chains were unaffected. F-actin was prepared from rabbit muscle according to the method of Spudich and Watt (27) and was dialyzed versus 10 mM MOPS (pH 7.0), 0.1 mM EGTA, 50 mM NaCl, 0.8 mM MgCl 2 , 0.2 mM ATP. MLCK (E (0.1%) ϭ 1.14) was prepared (28) with the modifications described (25) and was stored at Ϫ80°C in single aliquots with 10 mM DTT. 10 mM DTT was added to MLCK immediately after thawing and then stored on ice.
Thiophosphorylation of HMM-HMM was thiophosphorylated as described (29). This preparation is referred to as pHMM. The extent of thiophosphorylation was verified using 10% Tris-glycine gels (10 cm ϫ 10 cm, 12 lanes; Invitrogen) with standard Tris-glycine running buffer. This type of gel gave superior results to urea and/or urea-glycerol gels (30,31). The samples were precipitated with 3 volumes of cold acetone prior to the addition of sample buffer (8 M urea (ultrapure, Research Organics), 33 mM Tris-glycine (pH 8.6), 0.17 mM EDTA, 10 mM DTT (added immediately before use), bromphenol blue) to 6 -7 mg/ml protein. Approximately 25-30 g of HMM was applied to the gel. The samples were not heated above room temperature.
Peroxide Treatment of HMM and S1-HMM samples were thiophosphorylated where indicated and spun through a 5-ml buffer exchange column (32) prepared with Sephadex G-50 -80 resin (Sigma) in nonreducing buffer (10 mM MOPS, 50 mM NaCl (pH 7.0)). The samples (typically 1-2 mg/ml) were incubated with H 2 O 2 under specified conditions (see legends). To remove H 2 O 2 , the samples were either spun through buffer exchange columns to nonreducing buffer or treated with catalase, as indicated.
Single Turnover of HMM-MantATP in the Presence of Actin-This assay measures the rate of phosphate release from the acto-HMM MantADPP i state (25). Because phosphate release is rate-limiting, the decrease in fluorescence as MantADP dissociates from HMM can be used to measure phosphate release rates. The single-turnover assay allows populations of molecules or heads with different turnover rates to be elucidated. The assays were performed at 25°C in a temperature controlled stopped flow fluorimeter (Hi-Tech, SF-61DX2, Salisbury, UK) equipped with a 75-watt Xe-Hg lamp. The mixing dead time was ϳ2 ms. The excitation wavelength was 365 nm, and the excitation bandwidth was 4 nm. Emission was collected through a 389 low cut-off filter (Corion). For upHMM, formycin triphosphate was used instead of MantATP. The excitation wavelength was 313 nm, and the excitation bandwidth was 4 nm. Emission was collected through a 370 low cut-off filter (Corion). The experiment was done with two syringes. The first syringe contained 0. to the contents of the first syringe. The fluorophore binds maximally to pHMM in about 45 s (data not shown). After flushing the cuvette three times, 50 l was shot from each syringe (at 45 s), and the data were collected with the anti-bleaching shutter engaged for upHMM (1000 -1400 s; 1024 points) or without the shutter for shots under 60 s for pHMM and S1 (1024 points). All of the data were analyzed with Kaleidagraph software (Synergy Software, Reading, PA). The data were fit to a double exponential model (25). K ATPase (K m for actin) and V max for pHMM were determined by varying the actin concentration from 5 to 150 M and fitting the curve to the Michaelis-Menten equation as described (29).
Steady-state ATPase Assay-Steady-state [␥-32 P]ATP hydrolysis of S1 was measured in 10 mM MOPS (pH 7.0), 1 mM ATP, 2 mM MgCl 2, and 50 M F-actin at 25°C. Released 32 P i was separated from [␥-32 P]ATP with acidified, activated charcoal (33) and quantified by scintillation counting. The background [␥-32 P]ATP hydrolysis rates of actin alone and S1 in the absence of actin were subtracted.
Mass Spectrometry-UpS1 was treated with 1 mM H 2 O 2 for 30 min, alkylated with 10 mM iodoacetamide for 30 min, and subjected to nonreducing SDS-PAGE. The bands were stained with Coomassie Safe Stain, excised, reduced with DTT, treated with vinyl-pyridine, and subjected to in-gel trypsinolysis with modified sequencing grade trypsin (Promega). Following digestion, the samples were subjected to nanobore LC with on-line tandem mass spectrometry performed on a ThermoFinnigan LTQ-FT. This instrument is a hybrid of a linear ion trap with a Fourier transform-infrared cyclotron resonance detector (34,35). Searches for oxidative modifications of Cys and Met, alkylation of Cys, and N-acetyl Cys (ELC) were conducted with Mascot (Matrix Science).
Tracheal Smooth Muscle Experiments-See the supplemental materials and the legend for Fig. 9 for information regarding the tracheal smooth muscle experiments.

RESULTS
To evaluate the effects of H 2 O 2 on the ATPase kinetics of pHMM ( Fig. 1) and upHMM (Fig. 2), samples were heated for 30 min at 37°C in nonreducing buffer with H 2 O 2 or H 2 O 2 plus 5 mM EDTA. After rapidly removing H 2 O 2 , the transient kinetics of phosphate release was measured in the presence of 5 M actin. The data were fit to a double exponential model. This assay has two advantages over the more typical steady-state assay. First, it can monitor populations of molecules with different turnover rates, and second, it can monitor the total number of ATP-binding sites by analyzing the extent of fluorescence increase upon binding of a fluorescent ATP analog. The rate of the fast phase of turnover (as a percentage of an identically treated control without H 2 O 2 ) for pHMM is plotted versus H 2 O 2 concentration in Fig. 1A. Unheated controls (4°C) behaved essentially identically to heated controls (37°C exposure without H 2 O 2 , data not shown). The fast rate decreased to ϳ50%, and the slow rate increased to about 120% of control at 2.5 mM H 2 O 2 . An identical H 2 O 2 treatment in the presence of 1 mM MgADP (data not shown) gave a similar result, suggesting that nucleotide binding at the active site did not protect against the inactivation. Fig. 1B shows that the fast rate contributes ϳ80% and the slow rate contributes ϳ 20% of the total turnover amplitude prior to H 2 O 2 treatment. H 2 O 2 treatment caused the fractional amplitude of the fast and slow rate to decrease and increase, respectively. These data might suggest that untreated HMM contains ϳ20% oxidized heads or heads that behave like H 2 O 2 -oxidized heads. However, we have found that the highly similar nonmuscle IIB HMM (expressed in insect cells) (36) and the single head fragment S1 (see later) also show the 80% fast to 20% slow turnover behavior. This suggests that the biphasic turnover is an intrinsic property of smooth and nonmuscle myosin heads. The molecular basis of this phenomenon is under further investigation. The weighted rate plotted in Fig.  1C takes into account both rates and amplitudes and is indicative of the steady-state ATPase rate of the population of molecules that bind to nucleotide. In the absence of EDTA, the weighted rate decreases to ϳ40% of control (squares). Similarly, the total amplitude (Fig. 1D), which reflects the total number of nucleotide-binding sites, decreases to ϳ50% of control (squares). This was confirmed in a separate experiment moni- The data were fitted to a double exponential model. The fast rate is typically 80% of the total amplitude and has a rate of ϳ1.2-2.1 s Ϫ1 . The slow rate has a rate of ϳ0.04 -0.06 s Ϫ1 . A, rates of the fast (squares, no EDTA; circles, with EDTA) and the slow phases (diamonds, no EDTA; triangles, with EDTA) versus H 2 O 2 . The data are from two independent experiments. Each point was run in triplicate, and the data shown are the means and standard error of the six measurements. The turnover rate for the unheated control was 103 Ϯ 5% of the heated control. B, plot of the amplitudes (as percentages of total amplitude) of the fast (squares) and slow (circles) rates of turnover from the experiments (no EDTA) in A. C, the weighted rate equals (rate 1 )(amplitude 1 %) ϩ (rate 2 )(amplitude 2 %). No EDTA (squares) and with EDTA (circles) are for a single experiment with triplicate data points. An identical independent experiment gave similar results. Standard errors were calculated according to Ref. 59. D, total amplitude (percentage of control) versus H 2 O 2 for two independent experiments. No EDTA (squares) and with EDTA (circles). For all plots, the lines through the data are either a linear fit or interpolated. toring the rate and amplitude of MantATP binding under pseudo-first order conditions (data not shown). Only the amplitude and not the rate of binding was altered by H 2 O 2 . Therefore treatment with 2.5 mM peroxide results in two populations of heads, 50% with 40% activity and 50% with ϳ0 activity. If this sample had been measured by a steady-state assay, the activity would be 20% of control. In summary, H 2 O 2 affects the kinetics of pHMM in two ways. First, for those molecules that still bind to nucleotide, it decreases the steady-state ATPase by decreasing the rate and the fractional amplitude of a fast component and increasing the rate and fractional amplitude of a slow component. This is an effect upon V max , not K m . Actin titrations showed that the K ATPase (of K m for actin) of control pHMM was 83 Ϯ 18 M, and that of peroxide-treated pHMM was 80 Ϯ 21 M (data not shown). Second, H 2 O 2 causes ϳ50% of the molecules to either lose the ability to bind to nucleotide or drastically weaken their affinity for nucleotide.
The presence of EDTA during the H 2 O 2 treatment (5.0 mM) generally diminished the H 2 O 2 -induced inactivation of pHMM with respect to the fast rates and diminished the H 2 O 2 -induced activation with respect to the slow rates (Fig. 1A), as reflected in the weighted rates (Fig. 1C). EDTA also protected against the loss of ATP-binding sites (Fig. 1D), although the effect was not large.
The effect of H 2 O 2 on the kinetics of upHMM is shown in Fig. 2. The assay was the same as in Fig. 1, except that formycin triphosphate instead of MantATP was used as the fluorescent nucleotide. Fig. 2 (A and B) shows that the effect of H 2 O 2 is 2-fold. Without H 2 O 2 , the turnover data can be fit to a single exponential with a rate constant of ϳ0.0015 s Ϫ1 . H 2 O 2 causes this rate to increase slightly, and a new phase appears with a rate at about 10 times the control rate. The relative contribution of this faster rate increases with increasing H 2 O 2 concentration (Fig. 2B). Fig. 2C shows that H 2 O 2 increases the weighted rate by ϳ15-fold at 2 mM peroxide. Fig. 2D shows that treatment with 2 mM peroxide caused a loss of ϳ50% of the total ATPbinding sites as was found for pHMM (Fig. 1D). Peroxidetreated upHMM was capable of being phosphorylated (data not shown). As in pHMM, the effect of peroxide on upHMM was to alter the kinetics of a population of molecules that can still bind to nucleotide and to render about 50% of the molecules incapable of binding to nucleotide.
The effect of H 2 O 2 on the kinetics of upS1 is shown in Fig. 3. This subfragment has no tail and only a single head domain. It is unregulated in that both the unphosphorylated and phosphorylated forms have a high activity. The pattern of H 2 O 2 -induced kinetic effects is very similar to those observed for pHMM (Fig.  1). This is expected, as pHMM and upS1 have similar high catalytic activity and other similarities with respect to RLC structure (37). The observed changes in the two rate constants are similar, although the changes in amplitudes (Fig. 3B) are not as great. Again, ϳ50% of the total nucleotide-binding sites have been destroyed (Fig. 3D). Fig. 4 shows the protective effect of EDTA on the peroxidemediated inactivation of upS1 as measured with a steady-state ATPase assay. At 3 mM H 2 O 2 without EDTA, about 20% of the activity remains. This is consistent with the single-turnover data in Fig. 3, which predicts that at 2.5 mM H 2 O 2 the steady-state activity should be ϳ30% of control. EDTA (5 mM) protected against the inactivation, with about 70% of the activity remaining after a 3.0 mM H 2 O 2 treatment. These data are similar to those observed for pHMM (Fig. 1). Fig. 5 shows a time course of the reaction of upS1 with 3 mM H 2 O 2 . The percentage of inhibition of the steady-state ATPase activity correlated with the total number of thiols as measured by Ellman's reagent, DTNB (38). There is an initial rapid inactivation phase in which about two thiols are modified, followed by a slower phase in which approximately six or seven thiols (about one-half of the total) are modified after 30 min. At the  same time the ATPase was inhibited to ϳ70% of control. The tight correlation between the inactivation and the loss of reactive thiols suggests that oxidized thiols cause the ATPase inactivation. The final thiol oxidation states could be disulfides or higher oxidation states such as sulfenic or sulfonic acids. Fig. 6 shows the effect of treatment with the reducing agent, DTT, on H 2 O 2 -inhibited upS1. Treatment with DTT (50 mM) for 1 or 2 h caused reversal of the inhibition from 40% of control to ϳ66 and 77%, respectively, and suggests that the mechanism for at least some of the loss of ATPase activity is by thiol oxidation. Because it is known that DTT readily reduces disulfides and sulfenic acid(s) (R-SOH) (39) to thiols, it is likely that these modifications are responsible for some of the inhibited ATPase activity. Under these robust DTT treatment conditions the ATPase activity was not completely reactivated. This suggests that H 2 O 2 treatment causes some of the thiols to become oxidized further to DTT-unreactive states such as sulfinic (R-SO 2 H) or sulfonic acids (R-SO 3 H) or that methionine(s) is also oxidized. Fig. 7 shows the effects of DTT on the single-turnover kinetics of H 2 O 2 -treated pHMM. In this example the H 2 O 2 treatment inhibited the ATPase activity (of those heads that can still bind nucleotide) to ϳ30% of control (Fig. 7C). At the same time, ϳ54% of the heads lost the ability to bind to nucleotide and therefore have 0 activity (Fig. 7D). Treatment with up to 40 mM DTT did not increase the total ATP-binding sites (Fig. 7D) but did increase the weighted rate (Fig. 7C) to ϳ55% of control and partially reversed the pattern of kinetic rates (Fig. 7A) and amplitudes (Fig. 7B) characteristic of the H 2 O 2 -treated S1. These data suggest that DTT can partially restore the native kinetics of molecules still capable of binding nucleotide but has no effect upon the molecules that lost the ability to bind nucleotide. This suggests that the loss of ATP binding ability is not correlated to disulfide bond formation but perhaps to further thiol oxidation or to methionine oxidation (40).
To test for H 2 O 2 -induced intersubunit disulfide bond formation, we analyzed a nonreducing SDS gel of H 2 O 2 -treated upS1 (Fig. 8A). S1 prepared in this manner has most of the heavy chain internally cleaved into a ϳ65-kDa N-terminal fragment and a C-terminal 26-kDa fragment. A small amount of uncleaved heavy chain still remains at ϳ90 kDa. H 2 O 2 internally cross-linked the ELC, causing it to smear to higher R f . In addition, the 26-kDa heavy chain fragment was cross-linked to the 65-kDa heavy chain fragment (C-terminal heavy chain to N-terminal heavy chain). This latter cross-link was identified by excising, reducing, and subsequently electrophoresing the band onto a reducing gel (Fig. 8A, right lane). Mass spectral analysis of a tryptic digest of the ϳ90-kDa band identified heavy chain peptides from both the 65-and 26-kDa fragments (data not shown). The nonreducing gel in Fig. 8B shows that the presence of EDTA during H 2 O 2 treatment largely inhibits disulfide cross-linking of the 65/26-kDa peptide pair. Under these conditions EDTA does not fully protect against the loss of ATPase activity. Therefore peroxide-treated S1 can be partially inactivated without the above disulfide bonding, suggesting that the disulfide bond is not the only modification to the protein. Fig.  8C shows that the reductant in the sample buffer was sufficient to largely reverse the cross-link, as expected for a disulfide bond. Therefore, gel analysis revealed that there are at least two SH groups, one on the 65-kDa fragment and the other on the 26-kDa fragment that become disulfide-bonded (reversible by DTT) upon H 2 O 2 treatment and that EDTA can inhibit disulfide bond formation. Table 2 shows the results of a mass spectral analysis of H 2 O 2treated upS1. Listed are the peptides that were found to contain cysteine thiols completely oxidized to the sulfonic acid group . The aliquots were removed at the indicated times, and the reaction was terminated with catalase (250 units) followed by Sephadex G-25 gel filtration. The inhibition of the steady-state ATPase activity (solid line, left axis) relative to control activity (typically 130 -175 nmol of P i min Ϫ1 mg Ϫ1 ) and the moles of DTNB-reactive thiols (SH) per mol of S1 (dotted line, right axis) are plotted. For the DTNB assay, S1 was diluted to 0.67 mg/ml in 8 M urea, 50 mM Tris, pH 7.0, 10 mM EDTA, 0.1 M KCl and incubated at 25°C for 10 min prior to addition of DTNB to 1 mM from a 0.1 M stock made in 1 M Tris, pH 8.0. A 412 measurements were taken immediately. The ratio of DTNB reactive thiols per S1 was calculated using the extinction coefficient for the released anion of 14,140 M Ϫ1 cm Ϫ1 (38). The data points and error bars are the means and standard error, respectively, from three or four independent experiments.

FIGURE 6. Effect of treatment with the thiol-specific reducing compound DTT on H 2 O 2 -induced inhibition of upS1 steady-state ATPase activity.
After incubating upS1 (1.75 mg ml Ϫ1 ) with 0 (control) or 1 mM H 2 O 2 (30 min, 30°C), both reactions were terminated with catalase (250 units) and then were divided. One portion of each divided sample was then treated (25°C) with 0 or 50 mM DTT for 1 or 2 h, and the ATPase activity was measured. The data are the means Ϯ S.E. from four to seven independent experiments.
(cysteic acid). Sulfonic acids were not detected in the control. It is likely that one or more of these cysteine oxidations are responsible for the loss of tight ATP binding because sulfonic acids cannot be reduced by DTT. In all cases where a sulfonic acid was identified, corresponding peptides with carbamidomethyl-Cys (iodoacetamide-treated) and/or pyridylethyl-Cys (vinyl pyridine-treated) were also detected, indicating that only a fraction of S1 molecules were oxidized completely. Cys 121 , Cys 481 , Cys 524 , Cys 545 , and Cys 717 (SH1) were not reliably identified because of the inappropriate size and/or hydrophobicity of the tryptic fragments containing these residues. The remaining 14 of 18 total Cys in the S1 head and ELC were reliably identified (not listed in Table 2) and were either carbamidomethyl-Cys or pyridylethyl-Cys. With our experimental approach it should have been possible to identify Cys residues in the disulfide-linked 26 -68 kDa (Fig. 8) that obligatorily participated in disulfide bonds. These Cys should not react with iodoacetamide but should react with vinyl pyridine following reduction with DTT. However, for every tryptic fragment, peaks were detected with carbamidomethyl-Cys, indicating that these SDS-PAGE resolved disulfide-linked dimers contained populations of alternate disulfide linkages. All Cys in the ELC were reliably identified. For the disulfidelinked 26 -68-kDa band, it is also possible that two of the Cys in the myosin head, which were not identified by our mass spectral analysis, may have been responsible for the H 2 O 2 -induced disulfides. It is likely that Cys 717 in the 26-kDa fragment is involved because its alkylation can alter SMM regulation and ability to move actin in an in vitro assay (41). Fig. 9 shows the effects of H 2 O 2 treatment of intact porcine tracheal smooth muscle upon the rate and amplitude of force generation at pCa 9 ϩ 2.1 mM ATP following Triton X-100 permeabilization and RLC thiophosphorylation (see the supplemental materials for a representative trace of the entire protocol). H 2 O 2 caused a ϳ50% decrease in the amplitude but not in the rate of force generation. Note that the levels of thiophosphorylation were not affected by H 2 O 2 treatment (24). Because the effect of H 2 O 2 persists after All other samples were treated with DTT at the indicated concentrations for 10 min at 42°C after treating with and removing 2.5 mM H 2 O 2 and assayed for the rate of phosphate release as described in Fig. 1. A, rate values for the slow and fast phases are shown next to each bar. The statistics are similar to Fig. 1. B, percentage of total amplitude of the fast (top bar) and slow phases (bottom bar), respectively. C, the weighted rate (see Fig. 1 for definition) is plotted as a percentage of control (100%). The solid line is a fit to a hyperbola. D, the total amplitude (%).  FEBRUARY 16, 2007 • VOLUME 282 • NUMBER 7 permeabilization and complete RLC thiophosphorylation, this suggests that H 2 O 2 directly affects the force-generating complex. These data are consistent with a H 2 O 2 -induced loss of functional myosin heads. The fact that the rate of force generation is unaffected suggests that oxidation does not slow all myosin molecules but rather abolishes the activity of some myosin molecules. This is remarkably consistent with our finding that H 2 O 2 treatment disables some heads in regard to nucleotide binding in vitro. In the muscle (Fig. 9), DTT treatment at 10 mM for 10 min reversed the H 2 O 2 inhibition by ϳ50%. Further DTT treatment had little effect (data not shown). This suggests that DTT can reactivate a population of heads that were rendered incapable of force generation but does not rescue all heads. Again this is consistent with our in vitro finding that DTT cannot restore the heads that have lost the ability to bind to nucleotide (Fig. 7D).

DISCUSSION
The data in Figs. 1 and 2 demonstrate that the effect of H 2 O 2 upon the actin-activated ATPase kinetics of molecules that retain nucleotide binding affinity is to alter regulation, which is defined as the phosphorylated/unphosphorylated rate (Table  1). Treatment with 2.5 mM H 2 O 2 decreased the pHMM rate by 65% and increased the upHMM rate by 90%. There was no effect on K ATPase (K m ), only on V max . Regulation decreased from 800-to 27-fold under these conditions. Constructs of SMM with altered regulation typically have abnormally high ATPase activities in the unphosphorylated state and an abnormally low ATPase in the phosphorylated state (36,(42)(43)(44)(45)(46)(47). Alkylation of SMM thiols causes a similar effect (41,48,49) most likely attributed to reaction of SH1(Cys 717 ). The effects of H 2 O 2 on pHMM and upS1 were very similar in our studies, suggesting that they are not mediated by modification of the most reactive thiol on SMM, which is in the S2 region of the rod (Cys 892 ) (49) and which is not present in S1.
An important finding of this work is that H 2 O 2 causes a large population of the SMM heads to lose the ability to tightly bind to nucleotide. Myosin subfragments have been shown to contain variable relative amounts of weak ATP-binding heads (50) even under control conditions. However, we are unaware of any treatment known to cause loss of tight ATP binding prior to this work. It is known that an uncharacterized process can cause myosin to form ATP-resistant actomyosin complexes often termed "dead heads" (25,29). Indeed, ATP-unreactive heads must often be removed by actin selection prior to single molecule force and displacement measurements to allow the active heads to be observed. Skeletal myosin that is extensively modified with the thiol-selective reagent N-ethylmaleimide forms ATP-resistant actomyosin complexes (51), but it is not known whether this is due to the loss of tight ATP binding.
It is known that alkylation of SMM thiols can cause changes in the regulation of the ATPase activity of SMM (41, 48) similar to those observed here. However, because prior work measured ATPase activities by steady-state methods, it is not clear whether or not a population of the heads was rendered incapable of ATP binding.
We show that a robust DTT treatment cannot revive the ability to bind to ATP, suggesting that disulfide bonding is not the cause but rather further oxidized thiols to sulfonic acid or oxidation of methionines. Mass spectrometric analysis confirmed that seven total cysteine thiols, four on the heavy chain (Cys 753 , Cys 707 , Cys 388 , and Cys 582 ) and three on the ELC (Cys 31 , Cys 84 , and Cys 137 ) were oxidized to sulfonic acids. This was observed only for the H 2 O 2 -treated sample. Thiols are most likely initially oxidized to the unstable sulfenic acids (R-SOH) (52) and further oxidized to the sulfonic acids (R-SO3H). These data are consistent with a study of sulfenic acid formation in H 2 O 2 -perfused heart muscle (53). Sulfenic acid was detected in the myosin heavy chain after initial H 2 O 2 treatment but diminished as further oxidation proceeded, most likely because of conversion to sulfinic then sulfonic acids. Such oxidation occurred at ϳ10 -50-fold lower effective H 2 O 2 concentrations than used here, suggesting that the thiols in question are highly susceptible to oxidation. We also detected oxi-   Fig. 1 for definition. c Phosphorylated/unphosphorylated rate.
dized methionines but cannot assess the significance because they are commonly found in unoxidized samples digested from gels. Overall, the data suggest that the loss of nucleotide binding affinity is most likely due to an oxidation of one or more thiols to sulfonic acids. The location of the observed oxidized thiols ( Table 2) does not immediately suggest a mechanism for the loss of ATP binding ability. The ELC is not required for ATP binding. The locations of Cys 388 in the upper 50-kDa subdomain, Cys 582 in the lower 50-kDa subdomain, and Cys 753 in the converter domain do not appear to be critical to the ATP-binding site. Cys 582 is in loop 3 of the heavy chain. Loop 3 is known to be involved in actin binding in some myosin isoforms but is not known to be important to ATP binding affinity (54). Cys 707 is "SH2" in the SH1-SH2 helix, which when modified in other myosin isoforms inhibits the ATPase activity but is not known to abolish ATP binding. However, its oxidation is likely to affect the rearrangement of this important connector helix and could trap the motor in a conformation that could alter ATP binding. Note that the highly reactive SH1 (Cys 717 ) is also likely to be modified, but we were unable to detect this peptide in the mass spectrometry experiment. SH1 alkylation is known to affect the ATPase activity but does not abolish ATP binding for iodoacetamide-labeled skeletal myosin (55) nor for spin-labeled skeletal myosin (although the rate of binding was slowed) (56). Alternatively, these modification(s) collectively may be sufficient to partially unfold the myosin. Interestingly, skeletal myosin oxidized by peroxynitrite loses the ability to form the ADP-Vi complex in addition to partially unfolding (57). Further studies will be required to understand the mechanism of the observations from our study.
We have shown that the presence of EDTA can diminish the H 2 O 2 -induced alterations in the kinetics of pHMM (Fig.  1), upHMM (Fig. 2), and upS1 (Figs. 3 and 4). EDTA chelates trace transition metals in the buffer, and transition metals are known to increase the rates of thiol oxidation by two general mechanisms (58). Metal-thiol complexes are more nucleophilic than the respective protonated thiols and can therefore react faster with oxidants such as H 2 O 2 . Also, transition metals can activate and deliver H 2 O 2 to protein thiols, thus increasing the rate of thiol oxidation. Our data show that EDTA has a protective effect against H 2 O 2 -induced changes in kinetics, suggesting that thiol oxidation is a contributing factor.
Gel analysis revealed that there are at least two SH groups, one on the 65-kDa fragment and the other on the 26-kDa fragment, that become disulfide-bonded (reversible by DTT) upon H 2 O 2 treatment and that EDTA can inhibit disulfide bond formation. The SH1 helix, with SH1(Cys 717 ) and SH2(Cys 707 ), is in the 26-kDa fragment. It is likely that SH1 is involved in the disulfide bonding because it is known to be highly reactive. However, our mass spectral data did not reveal the location of this disulfide bond.
An analysis of the correlation between loss of reactive thiols and ATPase activity (Fig. 5) shows that there is a biphasic loss of thiols correlated to the loss of ATPase mediated by H 2 O 2 . This biphasic relationship is mirrored in thiol alkylation studies of SMM where the rapid inactivation phase was attributed to reaction of a single ELC or rod thiol (Cys 892 ). Our mass spectral data of upS1 (which does not contain Cys 892 on the rod) show that three thiols in the ELC are partially oxidized to sulfonic acids. Therefore ELC oxidation is most likely responsible for the rapid phase of the loss of ATPase activity. The slow phase most likely involves modification of Cys 717 (SH1) and the other thiols in Table 2.
An important question is whether or not it is possible that myosin oxidation is responsible for the observed H 2 O 2dependent decrease in maximal force generation in intact airway smooth muscle tissue ( Fig. 9 and supplemental data). Under these conditions, the level of myosin thiophosphorylation is the same for the control and H 2 O 2 -treated tissues. Here we correlate the in vitro kinetics obtained with purified SMM to the in situ kinetics of force development. H 2 O 2 -treated tissue has diminished amplitude of force development, but the rate is not altered (Fig. 9). DTT treatment only partially restored the amplitude. These properties are remarkably consistent with the kinetic properties of purified SMM. It appears that a population of the myosin heads in both the muscle and purified protein are rendered incompetent. In the tissue this is manifested as a decrease in maximal force generation and in the purified preparations as a loss of tight ATP binding for a population of the myosin heads. The fact that DTT partially restored the maximal force is consistent with the in vitro finding that DTT can restore those myosin heads that had retained ATP binding affinity. This interpretation requires that the heads with altered kinetics

cysteic acid) as determined by mass spectrometry of trypsin-digested upS1
Peptides from tryptic digest of upS1 following oxidation with 1 mM H 2 O 2 for 30 min at 37°C. Following peroxide treatment, cysteines were treated with iodoacetamide, and bands were resolved on SDS-PAGE, excised, then reduced with DTT, and treated with vinyl pyridine prior to trypsin digestion. Experimental M r is based on a search of b and y ions from the tandem mass spectrometry analysis. The masses listed are for the precursor ion prior to tandem mass spectrometry fragmentation.  FEBRUARY 16, 2007 • VOLUME 282 • NUMBER 7

Effect of Hydrogen Peroxide in Smooth Muscle Myosin
can generate force in the muscle at a rate similar to those in untreated muscle. We conclude that myosin oxidation is likely to be at least partially responsible for the altered contractile properties of airway smooth muscle exposed to H 2 O 2 . This may be important in diseased states in which H 2 O 2 is produced. Our findings have significance to previous work in the field in which similar levels of H 2 O 2 have been proposed to affect various cellular processes upstream of cross-bridge cycling in smooth muscles. Our study shows that oxidation of myosin alone may be partially responsible for the observations. In summary, we have shown that H 2 O 2 treatment of pHMM and upS1 causes a decrease in the ATPase activity, whereas for upHMM the activity is increased. Together these effects lead to a loss in phosphorylation-dependent regulatory properties ( Table 1). The changes in regulation are mediated by thiol oxidation, as evidenced by the inhibition of inactivation by EDTA, which can chelate heavy metals that enhance the oxidative power of H 2 O 2 ; by the partial reversal of the inhibition by DTT; and by the fact that we observed interdomain disulfide bonding and loss of DTNB-reactive thiols. A major finding is that a population of myosin heads lost the ability to bind nucleotide, and this effect could not be reversed with DTT. A comparison of the in vitro myosin kinetic data with the rate and amplitude of force development in airway smooth muscle tissue leads to the notion that myosin is oxidized by H 2 O 2 in situ, and the oxidation may be responsible for altering the muscle behavior.