Inactivation of the Inducible Nitric Oxide Synthase by Peroxynitrite

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Chem. Res. Toxicol. 1997, 10, 618-626

Inactivation of the Inducible Nitric Oxide Synthase by Peroxynitrite Andreas F. R. Hu¨hmer,† Clinton R. Nishida,‡ Paul R. Ortiz de Montellano,*,‡ and Christian Scho¨neich*,† Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045, and Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446 Received November 18, 1996X

The simultaneous production of superoxide and nitric oxide by stimulated human neutrophils leads to the formation of peroxynitrite, a physiologically important bactericidal agent. We have investigated two possible pathways for the inactivation of inducible nitric oxide synthase (NOS-II) by peroxynitrite: inactivation of NOS-II through oxidation of the tightly bound cofactor calmodulin (CaM) and direct interaction of ONOO-/ONOOH with the NOS-II protein. Studies of two model peptides indicated that the Ca2+-dependent binding to CaM of a typical highaffinity sequence, melittin, significantly prevented Met oxidation in CaM by ONOO-/ONOOH. In contrast, binding of the putative CaM-binding domain of human hepatocyte NOS-II (NOSII509-534) to CaM only marginally prevented the oxidation of Met residues in CaM. When the native NOS-II/CaM complex was exposed to peroxynitrite, CaM was inert toward oxidation. Nevertheless, even small amounts of peroxynitrite abolished the activity of NOS-II through direct interaction with the heme. The loss of activity was paralleled by a decrease in heme absorbance and a shift of the absorbance maximum from 419 to 409 nm. The presence of the cofactor tetrahydrobiopterin during peroxynitrite exposure did not prevent inactivation of the enzyme but altered the change of the heme spectrum, i.e., a shift of λmax from 419 to 420 nm rather than to 409 nm. In conclusion, peroxynitrite inactivates NOS-II through changes in the heme or its environment in NOS-II rather than via oxidation of the cofactor CaM. Much effort has been devoted to the investigation of the structure and regulation of nitric oxide synthases (NOS).1 The manifold functions of its product nitric oxide (NO•) in mammalian physiology suggest a highly differentiated regulation of the three isoforms of NOS (NOSI, neuronal; NOS-II, inducible; and NOS-III, endothelial). Consistent with their roles, the regulation and the assembly of the individual enzyme isoforms differ significantly. This difference is particularly evident for the Ca2+/CaM-dependent regulation of the NOS isoforms (13). For NOS-I, CaM modulates the intradomain electron transfer from NADPH to the flavin-binding domain and the interdomain electron transfer from the flavin-binding domain to the heme moiety (4), whereas for NOS-II such an effect of CaM is disputed (5). Binding of CaM and activation of NOS-I are Ca2+-dependent and can be inhibited by the addition of EGTA or the CaM antagonist trifluoperazin. In contrast, CaM binding to the inducible form of NOS-II is Ca2+-insensitive at intracellular Ca2+ concentrations. CaM binds very tightly (Kd e 1 nM) to a putative CaM-binding site of murine macrophage NOSII, residues 502-532 (6, 7), and resting levels of intracellular Ca2+ are sufficient for NOS-II stimulation (1). The inducible form of nitric oxide synthase is involved in the cytokine-induced production of NO• in vascular * Address correspondence to either of these authors. C. Scho¨neich: phone, (913) 864-4826; fax, (913) 864-5736. P. R. Ortiz de Montellano: phone, (415) 476-2903; fax, (415) 502-4728; e-mail, [email protected]. † The University of Kansas. ‡ University of California, San Francisco. X Abstract published in Advance ACS Abstracts, April 15, 1997. 1 Abbreviations: CaM, calmodulin; NOS-II, inducible nitric oxide synthase; NOS-I, neuronal nitric oxide synthase; NO•, nitric oxide; L-Arg, L-arginine; Met, methionine; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; H4B, (6R,6S)-2-amino-4-hydroxy-6-(Lerythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropteridine.

S0893-228x(96)00188-9 CCC: $14.00

smooth muscle cells, bronchial epithelial cells, and murine macrophages. Nitric oxide levels rise after 48 h postinduction and remain at a fairly high level for up to several days (8). Recently, compelling evidence for the presence of NOS-II in stimulated human neutrophils has been presented (9). Under inflammatory conditions, macrophages simultaneously produce superoxide (O2•-) and NO• (10), promoting the formation of peroxynitrite, ONOO- (11). Peroxynitrite is a very reactive but relatively selective oxidant that is currently receiving much attention because of its potential cytotoxic actions. It is now well established, using the presence of 3-nitrotyrosine as evidence, that peroxynitrite can form under conditions of oxidative stress in several tissues, including mouse lung alveolar phagocytic cells during influenza virus-induced pneumonia (12). The nitration of tyrosine is easily achieved by peroxynitrite (13, 14), but recent model studies have also demonstrated the nitrating potential of ClNO2, another nitric oxide/nitrite-derived species (15). Excess production of ONOO- not only affects the integrity of the surrounding tissue but may also contribute to modulation of NO• and/or ONOObiosynthesis. We have shown in vitro that ONOO-/ ONOOH rapidly oxidizes the Met residues of calmodulin (CaM), thereby reducing the ability of CaM to stimulate NOS-I (16). Moreover, CaM oxidation was observed in vitro during enzymatic NO• production at suboptimal levels of L-Arg. The production of peroxynitrite as a bactericidal agent during the action of macrophages is desirable. Thus, a negative feedback mechanism for NOS-II similar to that found for NOS-I involving the oxidation of CaM would © 1997 American Chemical Society

Inhibition of NOS-II by Peroxynitrite

appear to be disadvantageous. The tight binding of CaM to NOS-II can possibly prevent the oxidation of CaM during catalytic turnover. The stability of NOS-II toward peroxynitrite is an important issue since it has been shown that peroxynitrite can cross cell membranes (11, 19). Thus, peroxynitrite generated outside macrophages may diffuse across the cell membrane and reach targets inside the macrophage. We have investigated the possible inactivation of NOSII by peroxynitrite. Two potential pathways were examined as follows: (i) the oxidation of the tightly bound cofactor CaM and (ii) a direct interaction of ONOO-/ ONOOH with the NOS-II protein. CaM oxidation within the CaM/NOS-II complex did not occur, whereas CaM bound to two model peptides, melittin and a peptide comprising the putative binding sequence for CaM of human hepatocyte NOS-II (NOS-II509-534), underwent considerable oxidation. Nevertheless, NOS-II was readily inactivated by direct interaction of peroxynitrite with the heme moiety.

Experimental Section Materials. All chemicals used for the synthesis of peroxynitrite (see below) were obtained from Fisher Scientific and were of metal grade quality. Buffers and solutions used in the oxidation experiments were treated with Chelex 100 (Biorad; 5 g/100 mL) to minimize transition metal contamination. Trypsin was purchased from Worthington (Freehold, NJ). The peptide comprising the putative CaM-binding region of human hepatocyte NOS-II, NOS-II509-534 (NH2-KRRREIPLKL LVKAVLFACM LMRKTM-COOH) was obtained from Peptidogenic Research (Livermore, CA) and was of > 95% purity. The identity of the peptide was confirmed by MALDI-TOF mass spectrometry. All other chemicals and reagents were from Sigma (St. Louis, MO). Calmodulin purification, tryptic digestion, and HPLC analysis of the tryptic digest of CaM, as well as characterization of the tryptic fragments, were performed as described (16). Melittin, NH2-GIGAVLKVLT TGLPALISWI KRKRQQ-COOH (Sigma), was separated from contaminating phospholipase A2 by HPLC on a C-18 reversed phase column (Cobert, St. Louis, MO) using a linear gradient from 0% to 60% acetonitrile in aqueous 0.1% trifluoroacetic acid followed by a 30 min isocratic elution. The concentration of melittin was determined spectrophotometrically using the molar absorption coefficient 5470 M-1 cm-1 at 280 nm (20). Peroxynitrite Synthesis. Peroxynitrite that has a low ionic strength and is free of H2O2 was synthesized according to the procedure of Pryor et al. (17). Ozone generated from dry oxygen with a Welzbach Model T-23 ozonator (Philadelphia, PA) was purged through a 0.2 M sodium azide solution at pH 12. Caution: Ozone is a powerful oxidizing agent, is cytotoxic, and should be used with appropriate caution. To ensure complete reaction and to minimize residual azide in the peroxynitrite solution, the ozonation was continued for 100 min. The stock solutions were then purged with N2 in order to remove residual N2O and O3. Final peroxynitrite concentrations of ∼90 mM were obtained, as determined from its UV spectrum (λmax ) 302 nm, 302 ) 1670 M-1 cm-1) (18). The ONOO- stock solutions were then stored at -70 °C until used. Oxidation of CaM by Peroxynitrite in the Presence of a Target Sequence. CaM (60 µM in 10 mM Tris buffer, containing 1 mM CaCl2, pH 7.4), in the presence of either melittin or the NOS-II509-534 peptide at various molar peptide: CaM ratios, was exposed at 25 °C to different concentrations of peroxynitrite by the addition of small aliquots (0.5-3 µL) of the peroxynitrite stock solution. Control experiments were conducted as follows. First, a defined concentration of peroxynitrite was allowed to decompose in the reaction buffer (10 mM Tris buffer, 1 mM CaCl2, pH 7.4) before an aliquot of the peptide/ CaM complex was added to the reaction mixture (reverse order of addition). Subsequently, melittin or NOS-II509-534 was separated from CaM by the addition of 50 mM EGTA followed by

Chem. Res. Toxicol., Vol. 10, No. 5, 1997 619 several ultrafiltration and washing steps (50 mM Tris buffer, pH 8.1) through microconcentrators of 3 kDa cutoff (Amicon, Beverly, MA). The amount of CaM recovered after the ultrafiltration was calculated from the HPLC peak area of tryptic fragments T 1 and T 8. The corresponding peak areas of a tryptic digest of untreated CaM were used as standards. Typically, recoveries of 95 ( 8% of the starting material were achieved. Exposure of NOS-II to Peroxynitrite by Infusion. A 200 µL solution of 2 µM NOS-II in 100 mM Tris-HCl (pH 7.2) containing 10% glycerol was infused for short periods of time at 0.8-1.4 µL/min with an alkaline solution of 30 mM ONOOemploying a glass capillary-equipped syringe (see below). Though peroxynitrite is present in the stock solution in its anionic form (ONOO-), rapid equilibration with peroxynitrous acid (pKa ) 6.8) occurs in the reaction mixture:

H+ + ONOO- a ONOOH

(1)

The reaction of peroxynitrous acid with oxidizable substrates is generally faster than that of ONOO-, so it can be assumed that ONOOH is the major oxidant in the infusion experiments. The reaction mixture (200 µL) was slowly stirred (∼5-10 rpm) with a small magnetic stirring bar. After the infusion of ONOO-, an aliquot of the solution was used to determine the activity of NOS-II using the oxyhemoglobin assay (see below). The tetrahydrobiopterin (H4B) that was added to the NOS-II solution prior to ONOO- infusion was not stabilized in some of the experiments by addition of the usual 10-fold excess of dithiothreitol (DTT) to avoid competitive reactions of ONOO-/ ONOOH with DTT. In separate experiments we determined that H4B was relatively stable in 250 mM HEPES (pH 7.5) decomposing with a half-life of ∼30 min. For the analysis of CaM oxidation subsequent to an infusion of 2 µM NOS-II with a final concentration of 3.5 mM ONOOin 100 mM Tris-HCl (pH 7.2) containing 10% glycerol, the CaM was dissociated from NOS-II by adding 2 times the volume of 1.5 M K2HPO4 (pH 7.4) buffer containing 3.5 M NaCl and 200 mM EGTA. After incubation for g1 h at room temperature, the NOS-II was separated from the mixture by ultrafiltration with microconcentrators of 50 kDa cutoff (Amicon). The filtrate containing CaM was washed several times with water, and the volume was reduced by ultrafiltration with microconcentrators of 3 kDa cutoff to 50 µL. CaM was then subjected to tryptic digestion, and HPLC characterization of the tryptic digest was conducted as described elsewhere (16). In a control experiment, solutions containing 2 and 22.5 µM CaM alone were infused with 0.5 and 3.5 mM peroxynitrite, respectively, and the extent of CaM oxidation was monitored. For these experiments it was important to determine whether CaM was recovered quantitatively from the complex with NOSII. A solution of a defined concentration of native CaM which had not been treated by ultracentrifugation was subjected to tryptic digestion, and the tryptic digest was analyzed by HPLC. Two fragments of the tryptic digest, denoted as T 1 and T 8, have no Met residues and are not oxidized. The peak areas of these fragments have been taken as a reference for all other quantitative measurements. Thus, solutions of either 2 or 22.5 µM CaM and 2 µM NOS-II which had been exposed to peroxynitrite and ultracentrifugation (see above) were subjected to tryptic digestion and HPLC analysis. Based on the peak areas of T 1 and T 8, respectively, it appears that we recovered 7986% of the CaM (Table 1, column 4). The multiple ultracentrifugation steps probably account for the small loss of CaM. Importantly, the recoveries of native CaM and CaM from the NOS-II complex were similar, indicating that our method recovered CaM quantitatively from the NOS-II complex. All experiments involving addition of peroxynitrite were compared to a reverse order of addition control experiment. Infusion Device and Control of the Amount of Peroxynitrite Delivered. A 500 µL luer tip gas-tight syringe (Hamilton, Reno, NV) was connected to a plastic adapter and a 1/ in. ZDV Union Peek with a finger-tight HPLC fitting. The 8 outlet side of the HPLC fitting was a peek tubing of 0.02 in. i.d. (orange color code from Upchurch Scientific, Oak Harbor, WA)

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Hu¨ hmer et al.

Table 1. Oxidation of CaM Bound to NOS-II or CaM Alone Exposed to Different Concentrations of ONOO-/ONOOHa NOS-II (µM)

CaM (µM)

state of CaM

ONOO(µM)

ONOO- delivery method

CaM recovery (%)

sulfoxide formation (%)

2 2 2

2 2 2 2 2 22.5

bound to NOS-II bound to NOS-II bound to NOS-II unbound unbound unbound

500 500 3500 500 3500 3500

addition reverse order of addition infusion infusion infusion infusion

79 ( 16 81 ( 17 84 ( 21 98 ( 5 96 ( 5 86 ( 12

NDb NDb NDb 53 ( 5 61 ( 4 30 ( 9

a After exposure, CaM was isolated and subjected to tryptic digestion as described in the Experimental Section. HPLC characterization of the tryptic map yielded the quantitative recovery of CaM (column 6) and the extent of Met sulfoxide formation in CaM (column 7). The experimental details for calculation of the recovery of CaM are described in the Experimental Section. The percentage of sulfoxide formation was calculated by dividing the peak area from the individual sulfoxide-containing fragment by the sum of the peak areas of the parent peak and all chromatographic peaks derived from it. b ND ) not detectable.

in which a glass capillary with 50 µm i.d. (Polymicro Technologies, Phoenix, AZ) was inserted. The syringe was mounted on an ambulatory infusion pump, Model MS 26 (Graseby Medical, Atlanta, GA), and the delivery rate was set to 99 mm/day. The syringe was only used between the 300 and 100 µL marks of the syringe to ensure constant delivery throughout the course of the experiment. Typically, the delivery rate was 0.8-1.4 µL min-1 when a 30 mM ONOO- stock solution was used. In separate experiments the amount of peroxynitrite delivered to the reaction mixtures was determined. A volume of the peroxynitrite stock solution identical to that delivered in an actual infusion experiment was infused into 200 µL of 1 M NaOH, and the concentration of ONOO- in the final volume was determined spectrophotometrically at λ ) 302 nm using ) 1670 M-1 cm-1 (21). This value was compared to the expected value, calculated on the basis of the total infusion volume of the ONOO- stock solution. Assay of NOS-II Activity. The activity of NOS-II was measured by monitoring the production of NO• employing the hemoglobin assay essentially as described by Murphy and Noack (22). Briefly, in a total volume of 500 µL of 50 mM HEPES buffer (pH 7.5) containing 0.4 mM DTT, 0.1 mg/mL bovine serum albumin, 10 µg/mL catalase, 10 µM oxyhemoglobin, 20 µM H4B, 500 µM L-Arg, 200 µM NADPH, and 5 µg of NOS-II, the production of NO• was initiated by the addition of NADPH. The rate of NO• production at 37 °C was followed by monitoring methemoglobin production at λ ) 401 nm (the isosbestic point at λ ) 411 nm was used as the reference point) on a Varian Cary multiwavelength spectrophotometer. The specific activity of NOS-II was calculated from the absorbance change using ∆401 ) 60 000 M-1 cm-1. Separate control experiments were conducted to detect any interference or artifacts caused by residual azide and/or peroxynitrite degradation products. The activity of NOS-II was determined under optimum conditions (maximum activity) throughout the experiments to guarantee day to day reproducibility. DNA Manipulation. NOS-II mouse macrophage cDNA was kindly provided by Stephen Black (UCSF). A 5′-terminal NdeI site and a 3′-terminal XbaI site were introduced into pGem/ NOS-II using the Transformer Site-Directed Mutagenesis Kit (Clontech). The mutagenic oligonucleotide primer 5′-GCAAAG TCTCAC ATATGG CTTGCC CC was used to create the NdeI site immediately preceding the starting Met codon and the primer 5′-GGCTCT GACAGT CTAGAG TTCCAG C to create the XbaI site immediately following the termination codon. The primer 5′-GTTTCT TAGACC TCGAGT GGCACT TT was used to convert the unique AatII site in pGem to a unique XhoI site to allow for selection of the mutagenized strand. The NdeI/XbaINOS-II gene was then subcloned into pcWori/6xHis expression plasmid, which contains immediately upstream of the NdeI site a sequence coding for a 6-histidine N-terminal tail to facilitate purification of the expressed protein. Human CaM cDNA was kindly provided by Emanuel E. Strehler (Mayo Clinic). Shunsuke Ishii (Institute of Physical and Chemical Research, Tsukuba, Japan) generously provided pT-GroE, a pACYC-derived plasmid containing a chloramphenicol resistance gene, a T7 promoter, and the p15A origin of replication that allows cotransfection with ColE1 ori-containing plasmids. 5′-NdeI and 3′-BamHI sites were created in the CaM

gene using polymerase chain reaction (PCR) cycling, Vent polymerase (New England Biolabs), and the primers 5′GGGGCG TCCATA TGGCTG ACCAGC TG, which creates the NdeI site, and 5′-CGGCCG GATCCT CACTTT GCAGTC ATC, which creates the BamHI site. The CaM gene was then subcloned into pT-GroE, resulting in the loss of the NdeI/ BamHI-cloned GroES and GroEL genes and producing CaMexpression plasmid pT7/CaM. NOS-II Expression and Purification. Expression of active NOS-II in Escherichia coli required the coexpression of CaM. BL21 (DE3) cells were cotransformed with pcWori/NOS-II and pT7/CaM. Cultures (1.5 L) were incubated at 37 °C to an OD600 of 0.8-1.0, at which point IPTG (0.8 mM) was added. The cultures were then cooled and incubated at 22 °C for an additional 16-20 h. Cells were harvested and kept at -70 °C until purification. The protein was purified by affinity chromatography. Cells were suspended in lysis buffer A (50 mM Tris-HCl, pH 7.8, 10% glycerol, 200 mM NaCl, 5 mM β-mercaptoethanol, 2 µM hemin, 2 µM FAD, 2 µM FMN, 0.1 mM PMSF, 1 µM leupeptin, 1 µM pepstatin, 1 µg/mL antipain), sonicated, and centrifuged for 30 min at 30 000 rpm to yield crude lysate. After addition of 5 mM imidazole to the lysate, it was applied to a Ni-NTA (Novagen) metal affinity column to bind the 6xHis-containing protein. Washing was done with 5-10 column volumes of buffer A (except with 500 mM NaCl) plus 5 mM imidazole. Semipure protein was eluted with 100 mM imidazole in buffer A and applied to a 2′,5′-ADP-agarose column. Washing was done with at least 10 column volumes of buffer B (50 mM HEPES, pH 7.5, 10% glycerol, 300 mM NaCl, 5 mM β-mercaptoethanol, 2 µM hemin, 2 µM FAD, 2 µM FMN, 0.1 mM PMSF, 1 µM leupeptin, 1 µM pepstatin, 1 µg/mL antipain) followed by 5 column volumes of buffer C (buffer B minus hemin, FAD, FMN, and the protease inhibitors). Final pure protein was obtained by elution with buffer C (except with 500 mM NaCl) plus 80 mM AMP (20 mM 2′-AMP and 60 mM 3′-AMP; only 2′-AMP is functional in eluting bound protein).

Results Oxidation of CaM by Peroxynitrite in the Presence of Melittin or NOS-II509-534. We have previously shown that exposure of CaM to ONOO-/ONOOH results in the exclusive oxidation of Met to Met sulfoxide (16). In the present work, solutions of 60 µM CaM in 10 mM Tris-HCl (pH 7.4) containing 1 mM CaCl2 and melittin at CaM:melittin molar ratios of 1:0 (control), 1:0.5, 1:1, and 1:2 were exposed to ONOO- at a 5-fold molar excess of ONOO-/ONOOH over CaM. Subsequent isolation and characterization of the CaM by tryptic digestion and HPLC analysis of the tryptic fragments revealed that the Ca2+-dependent binding of melittin protected the Met residues of CaM from oxidation by ONOO-/ONOOH (Figure 1A). In the absence of a complementary binding peptide but in the presence of Ca2+, nearly 34 ( 8% of all nine Met residues of CaM were oxidized to their sulfoxides, with the C-terminal Met residues, designated


Chem. Res. Toxicol., Vol. 10, No. 5, 1997 621

Figure 1. Protection of CaM Met residues by melittin and a NOS-II consensus CaM-binding sequence. Solutions of 60 µM CaM in 10 mM Tris buffer (pH 7.4) contained 1 mM CaCl2 and different molar ratios of (A) melittin (NH2-GIGAVLKVLT TGLPALISWI KRKRQQ-COOH) or (B) NOS-II509-534 (NH2-KRRREIPLKL LVKAVLFACM LMRKTM-COOH). The systems were exposed to a 5-fold molar excess of ONOO- over CaM. Met residue-containing tryptic fragments were designated as T 4 (Met36), T 5 (Met51,71,72), T 7 (Met76), T 12 (Met109,124), and T 13 (Met144,145). Bars indicate the extent of Met oxidation in the respective tryptic fragments as quantitated by HPLC and are the means ( SE of three independent oxidation experiments. The percentage of sulfoxide formation was calculated by dividing the peak areas of the individual sulfoxide-containing fragments by the sum of the peak areas of the parent peak and all the chromatographic peaks derived from it.


as tryptic fragments T 13 (Met144 and Met145) and T 12 (Met109 and Met124), being the most susceptible to oxidation (Figure 1A, panel I). Met oxidation in CaM Cterminal tryptic fragments T 13 and T 12 and N-terminal fragments T 7 (Met76) and T 4 (Met36) progressively decreased as the melittin:CaM molar ratio rose from 0:1 to 1:1 (Figure 1A, panels I and III). No further significant changes in the oxidation pattern were observed when the melittin:CaM molar ratio increased to 2:1 (Figure 1A, panel IV). Overall, the oxidation of N-terminal tryptic fragment T 5 (Met51, Met71, and Met72) appeared to be little affected by melittin. In the presence of 2 mM EGTA there was little oxidation of the tryptic fragments T 4, T 5, and T 7, whereas the Met residues of tryptic fragments T 12 and T 13 were still significantly oxidized (Figure 1A, panel V). A control experiment in which CaM was exposed to peroxynitrite in the presence of EGTA but in the absence of melittin (data not shown) gave data essentially identical to that in Figure 1A, panel V. Generally, the complexation of Ca2+ by EGTA inhibits the Ca2+-dependent conformational changes of CaM as well as the binding of melittin to CaM. Under such conditions two parameters determine the susceptibility of the CaM Met residues to oxidation by ONOO-/ ONOOH: (i) the different exposure of the Met residues in apoCaM versus Ca2+-saturated CaM and (ii) the competition between CaM and melittin for reaction with ONOO-/ONOOH. The only amino acid within melittin sensitive to peroxynitrite modification is a tryptophan (Trp). A comparison of the respective rate constants for the reaction of peroxynitrous acid with the free amino acid Trp, kTrp+ONOOH ) 184 M-1 s-1 (23), the free amino acid Met, kMet+ONOOH ) 902 M-1 s-1 (24), and CaM, kCaM+ONOO-/ONOOH,pH7.4 ∼8000 M-1 s-1 (16), reveals that a Trp does not compete effectively for ONOO-/ONOOH in the presence of an equimolar concentration of CaM (CaM has nine Met residues). Thus, the overall decrease in CaM oxidation in the presence of EGTA is best rationalized by conformational changes which result in the Met residues of Ca2+-deficient CaM being less surface exposed than those of Ca2+-saturated CaM. It is to be noted that the extent of Met sulfoxide formation from 60 µM CaM is less than that for 240 µM CaM when the CaM:ONOO-/ ONOOH ratio is 1:5 (16). This is readily explained by the fast competitive decomposition of peroxynitrite to nitrate, which results in less efficient protein oxidation at lower CaM concentrations. The protective effect of NOS-II509-534 was less significant than that of melittin and affected mostly tryptic fragment T 13 (Figure 1B). For example, a 1:1 molar ratio of CaM:NOS-II509-534 afforded less than 50% protection from oxidation of T 13, compared to ∼70% protection of T 13 in the presence of melittin. Even more pronounced was the difference for T 12, for which the presence of melittin gave ∼60% protection but NOSII509-534 only ∼25%. The oxidation of the tryptic fragments T 4, T 5, and T 7 was little affected by the presence of NOS-II509-534. More insight into the mechanism of how NOS-II509-534 protects the C-terminal Met residues of CaM from oxidation was obtained from a comparison of panels III-V of Figure 1B. If protection were merely through the Ca2+-dependent binding of NOS-II509-534 to CaM (as in the melittin:CaM system), then protection should be more pronounced in the presence of Ca2+, i.e., absence of EGTA. Comparison of Figure 1B, panels III and V, reveals a much smaller difference in the protection of CaM by NOS-II509-534 versus melittin in experiments conducted in the presence and absence of Ca2+ (Figure

Hu¨ hmer et al.

1A, panels III and V). Therefore, we conclude that complexation is not a major pathway of protection of CaM by NOS-II509-534 and that protection is afforded by simple chemical competition for peroxynitrite. In agreement with this, if NOS-II509-534 protects the CaM Met residues simply by competitively reacting with ONOO-/ONOOH, we would expect better protection at CaM:NOS-II509-534 ratios > 1:1, i.e., 1:2 (Figure 1B, panel IV). This is indeed the case, particularly for T 12 and T 13. NOS-II509-534 contains one cysteine (Cys) and three Met residues that may compete for reaction with peroxynitrite. On the basis of the rate constant for the oxidation of a proteinbound Cys by ONOO-/ONOOH at pH 7.4, k ) 2600 M-1 s-1 (25), the Cys residue of NOS-II509-534 should be an important target for peroxynitrite. Consistent with this, MALDI-TOF mass spectrometric analysis of a peroxynitrite-exposed NOS-II509-534/CaM mixture revealed significant formation of covalently bound NOS-II509-534 dimers. Exposure of NOS-II to High Concentrations of Peroxynitrite: Analysis of CaM Oxidation. To determine the extent of CaM oxidation after exposure of the complete NOS-II/CaM system to peroxynitrite, NOSII was exposed to a large excess of ONOO-/ONOOH. The recombinantly expressed NOS-II contained a 6xHis tag to assist in purification of the protein. We do not expect any influence of the His tag on the experiments because a reaction of peroxynitrite with His has not been documented. A 2 µM NOS-II/CaM solution in 200 µL of 100 mM Tris-HCl (pH 7.2) was exposed to various concentrations of peroxynitrite either by slowly infusing a stock solution of the oxidant for up to 20 min with the total exposure of NOS-II adjusted to e3.5 mM peroxynitrite or by addition of a bolus of peroxynitrite stock solution with the final concentration adjusted to 500 µM ONOO-/ ONOOH (Table 1). After separation and isolation of the CaM subunit from NOS-II by treatment with high salt concentrations, tryptic digestion of the protein revealed that CaM survived completely unoxidized even at high concentrations of ONOO-/ONOOH. In a control experiment it was shown that exposure of 22.5 µM native CaM by itself to ONOO-/ONOOH under the same conditions resulted in the oxidation of 30 ( 9% of all nine Met residues. Exposure of NOS-II to Low Amounts of Peroxynitrite by Infusion: Activity of NOS-II. Through the use of a specially equipped syringe mounted on an infusion pump, we were able to simulate the exposure of NOS-II to low steady state concentrations of ONOO- over periods of 20 min or longer. The efficiency of the conversion of NO• into peroxynitrite in vivo is not known but depends, among other things, on the endogenous concentrations of superoxide and heme proteins. If we assume that ca. 75% of NO• is converted into ONOO-, we can use the specific activity of NOS-II to calculate a hypothetical flux of peroxynitrite to which our NOS-II system might be exposed in vitro to mimic the in vivo situation. Our NOS-II preparations had a specific activity of 550 ( 75 nmol of NO• min-1 mg-1, so that 50 µg of NOS-II would yield 27.5 ( 3.8 nmol of NO• min-1. On the basis of this assumption, we exposed the enzyme to between 14 and 29 nmol min-1 peroxynitrite, which corresponds to 70-140 µM min-1. The infusion of protein with ONOO-/ONOOH was terminated at different time intervals, and the activity of NOS-II was determined under optimum conditions. Our experiments showed that only small amounts of ONOO-/ONOOH were needed to substantially decrease the NOS-II activ-


Figure 2. Influence of H4B on the inactivation of NOS-II by peroxynitrite. NOS-II was exposed to low levels of ONOO- by infusing approximately 20 nmol min-1 ONOO- into 200 µL of 100 mM Tris buffer (pH 7.2) containing 10% glycerol and 2 µM NOS-II. One set of experiments also contained 2 µM H4B (b), while a second set of experiments was conducted in the absence of H4B (]). Aliquots from these infusion experiments were used to determine the NOS-II activity (y-axis) using the oxyhemoglobin assay. All activity measurements involving peroxynitrite were corrected for the respective reverse order of addition experiment, the results for which are shown as a separate line (+). The lower trace (9) indicates the measured activity after H2O2 addition to NOS-II. The abscissas indicate (i) the final concentration of ONOO- or H2O2 that was achieved over the course of the experiment (bottom x-axis) and (ii) the time that the NOS-II was infused with ONOO- at an average ( SE flow rate of 0.89 ( 0.13 µL min-1 (top x-axis).

ity. When the NOS-II was exposed to ONOO-/ONOOH for 2.5 min at a flux of ∼20 nmol of ONOO- min-1 (e.g., to a total concentration of 240 µM ONOO-/ONOOH), 50% of the maximum activity was lost when compared to a control incubation without the peroxynitrite. The final molar ratio of NOS-II to ONOO-/ONOOH in this experiment was 1:125. Infusion for more than 8 min (corresponding to >770 µM ONOO-/ONOOH; NOS-II:ONOO-/ ONOOH < 1:400) completely abolished the NOS-II activity. To distinguish the effects of ONOO-/ONOOH on the monomeric form from those on the NOS-II dimer, we performed the experiment in the absence and presence of added tetrahydrobiopterin (H4B). The loss of activity caused by ONOO-/ONOOH was neither delayed nor reduced when 2 µM H4B was added to 2 µM NOS-II prior to addition of the peroxynitrite (see Figure 2). Control experiments (reverse order of addition) indicated that peroxynitrite degradation products had a significantly smaller effect on NOS-II activity (Figure 2, upper trace). In separate experiments we determined that the degradation products NO2- (400 µM) and NO3- (800 µM), as well as experimental factors such as temperature and mechanical stirring, reduced the NOS-II activity to 60% of its original value within 20 min (∼2% min-1 activity loss in the infusion experiment). Spectral Changes of NOS-II Caused by Exposure to Peroxynitrite. Exposure of NOS-II to ONOO-/ ONOOH caused a time-dependent loss of heme absorbance concomitant with a blue shift of the absorbance maximum from 419 to 409 nm. These spectroscopic changes were paralleled by loss of the activity of NOSII. A small decrease in the Soret absorbance maximum (Figure 3) that was accompanied by loss of activity (Figure 2, upper trace) was also observed in the control


Figure 3. Absorbance spectrum of NOS-II as a function of exposure to peroxynitrite. The absorbance spectrum of NOS-II (8 µM) in 100 mM Tris-HCl (pH 7.2) containing 10% glycerol exposed to different amounts of ONOO- was recorded and compared to a control experiment. The trace at the bottom is a spectrum from NOS-II that was exposed to 750 µM H2O2. All spectra were aligned according to their absorbance at 700 nm except the lowest spectrum which was shifted downward to preserve clarity.

Figure 4. Absorbance spectrum of NOS-II as a function of exposure to peroxynitrite in the presence of H4B. The absorbance spectrum of NOS-II (2 µM) in 100 mM Tris-HCl (pH 7.2) containing 10% glycerol exposed to different concentrations of ONOO- in the presence of 2 µM H4B was recorded.

experiments. This is due to experimental factors, such as decomposition products, temperature, and mechanical stirring. However, no blue shift in the Soret maximum in the control experiments was detected. Comparable spectroscopic changes (Figure 3), accompanied by a loss of NOS-II activity (Figure 2), were observed when H2O2 was added to NOS-II (Figure 3). The spectroscopic changes induced by ONOO-/ONOOH were different when 2 µM H4B was added prior to the infusion. Here, though NOS-II activity was also lost, the absorbance maximum underwent a slight red shift from 419 to about 420 nm (Figure 4). Mouse NOS-II purified from macrophages, as well as mouse and human recombinant NOS-II purified in the presence of H4B and L-Arg, exhibit a typical high-spin spectrum with a Soret maximum at ∼ 400 nm. Recombinant mouse NOS-II purified in the absence of H4B and L-Arg normally exhibits a typical low-spin spectrum which can be converted to the normal high-spin spectrum by the addition of exogenous


H4B and L-Arg. Identical absorbance changes were observed when either H4B alone or H4B plus DTT was present during the exposure. Furthermore, the addition of DTT or H4B to the cuvette after the infusion experiment did not restore the typical NOS-II spectra of the H4B-free and H4B-bound forms with maxima at 419 and 400 nm, respectively.

Discussion We now know that CaM constitutes a biological target for oxidation in vivo. For example, Met residues of CaM isolated from aged rat brain were significantly oxidized to Met sulfoxide, a finding paralleled by a reduced ability of CaM to stimulate target enzymes such as the plasma membrane Ca2+-ATPases of synaptic vesicles and erythrocytes (26). In the present paper we wanted to determine (i) whether complexation of CaM to its target enzyme(s) prevents such oxidative modifications and (ii) if there is a peroxynitrite-dependent inactivation of NOSII through oxidation either of CaM or the NOS-II protein. CaM is a tightly bound subunit of NOS-II, and this tight binding may be necessary to prevent modification of the CaM by peroxynitrite during the action of macrophages. In parallel with our investigation of NOS-II, we conducted model studies in which CaM was bound to both melittin and a peptide (NOS-II509-534) comprising the putative CaM-binding domain of human hepatocyte NOSII. CaM binds with melittin with a very high affinity in the presence of Ca2+ and forms a 1:1 complex (Kd ) 3 nM) (27). Melittin does not contain any major targets for oxidation by ONOO-/ONOOH except for a single Trp residue whose rate constant for oxidation by ONOO-/ ONOOH is significantly lower than that for the oxidation of Met in CaM (cf. kONOOH+Trp ) 184 M-1 s-1 (23); (kONOO-1 s-1 (16)) and was therefore ideal +CaM, pH 7.4 ∼ 8000 M for the present study. The Met residues at both terminii of CaM play a significant role in the interaction between the target sequence(s) and CaM (28, 29). It was shown that modification of these Met residues leads to loss of binding and/or loss of the stimulatory action of CaM (16, 30). Complex formation between CaM and melittin induces the bending of CaM and facilitates Ca2+ uptake. This conformational alteration directs the Met residues of both terminal lobes of CaM into complementary binding pockets in the target sequence, producing a strong hydrophobic interaction between the two proteins. When bound, oxidants such as ONOO-/ONOOH should have little access to the Met residues in CaM. However, this protective effect appears to depend on the affinity of the individual target sequence binding sites for the individual binding domains of CaM. NMR studies of CaM/melittin complexes indicate that the amino terminus of CaM constitutes a low-affinity binding site that may be less involved in melittin binding (31). This rationalizes the lower protection of T 5 but not the better protection of T 4 in our experiments. As we know that the oxidation of a single Met residue of CaM changes its binding to a target protein, e.g., to erythrocyte plasma Ca2+-ATPase (32), a further complication is that a single oxidative event may change the structure of the melittin/ CaM complex and expose additional Met residues to oxidation. Nevertheless, we can demonstrate that a strong interaction such as binding of a target peptide to CaM can reduce ONOO-/ONOOH-mediated Met sulfoxidation. The complexation of CaM with NOS-II509-534 only led to some protection of the C-terminal domain of CaM,

Hu¨ hmer et al.

whereas not even the Met36-containing N-terminal fragment T 4 was protected. Moreover, allowance must be made for the fact that protection is not through binding alone but also through direct competitive reaction of ONOO-/ONOOH with NOS-II 509-534 instead of CaM. The isolation of significant amounts of a covalently linked NOS-II509-534 dimer confirms that ONOO-/ONOOH indeed reacts with NOS-II509-534, presumably via thiol oxidation and disulfide formation. Since the rate constants for the reactions of ONOO-/ONOOH with a protein-bound Cys (k ) 2600 M-1 s-1 at pH 7.4) (25) and free CaM (k ∼ 8000 M-1 s-1) (16) are not too different, and since not all Met residues of CaM are expected to be fully accessible in the complex, we suspect that the apparent protection of the oxidation of the CaM fragments T 12 and T 13 is not through complexation but simply through competition. This would also explain some protection of, for example, T 12 and T 13 by NOSII509-534 for NOS-II509-534:CaM ratios > 1:1 under conditions where the protection exerted by melittin leveled off. Thus, whereas melittin is an appropriate model peptide to investigate binding-mediated protection of CaM, NOSII509-534 appears to be an inappropriate model system for such studies. The result with NOS-II509-534 not only is different from that with melittin but also places in question the strong interaction between CaM and the NOS-II peptide suggested by others (3, 7, 33). Several possible rationales may be considered as follows. (i) The putative binding site of CaM does not account for the complete binding of CaM. As in the γ-subunit of phosphorylase kinase (34) and the adenylate cyclase of Bordella pertussis (7), NOS-II may have a second, as yet unidentified, binding site that contributes to the binding of CaM. Nathan and co-workers (35) recently provided evidence that residues in the carboxyl-terminal half of NOS-II, in addition to the consensus CaM-binding sequence, are required for Ca2+-independent CaM binding. In this respect, the three-dimensional structure of the NOS-II domain surrounding the CaM-binding site plays an important role in the binding of CaM to NOS-II. It has been shown that CaM binding is not responsible for the dimeric structure of the protein but is necessary for the structural integrity of the enzyme (2). (ii) The tight Ca2+-dependent binding of CaM is only valid for the high-affinity binding site of CaM. This could explain our observation that only the C-terminal Met residues in CaM are partly protected from oxidation by NOS-II509-534. The binding of CaM in the presence of Ca2+ to NOS-II503-532, the putative CaM-binding site of the murine macrophage enzyme (7), has been reported by three different groups using two different approaches and several detection methods (3, 7, 33). However, contradictory results were reported on the binding of CaM in the presence of EGTA. Using surface plasmon resonance, Zoche et al. showed binding of CaM to murine macrophage NOS-II503-532 both in the presence of Ca2+ and in the presence of low amounts of EDTA (3.4 mM) (33). Low amounts of Ca2+ or 2 mM EGTA in the incubation buffer were sufficient for the formation of a complex as detected by a gel shift assay (7). The reported formation of multiple bands and the lack of complex formation in the presence of 2 mM EGTA using the same gel shift assay (but a different detection method) (3) might reflect dissociation of CaM from the binding sequence due to a high urea concentration in the gel shift assay (36). (iii) Interactions other than hydrophobic association play a role in CaM binding. Recently, Fosetta et al. (2)


provided evidence that CaM binds to NOS-II in a similar manner as it does to phosphorylase kinase, where electrostatic interactions play a role in the binding of CaM to the target sequence. They were able to dissociate CaM from NOS-II by a combination of calcium chelators and high salt concentrations. Using similar conditions we were able to separate CaM from NOS-II for characterization of its integrity after exposure to ONOO-/ ONOOH. When the whole NOS-II enzyme complex, including tightly bound CaM, was subjected to high concentrations of ONOO-/ONOOH, no modification of the CaM Met residues was detected. However, in a control experiment in which CaM alone was exposed to ONOO-/ONOOH, significant Met oxidation occurred. Thus, a structurally intact NOS-II/CaM complex completely protects CaM from oxidation, whereas the putative CaM-binding domain of NOS-II does so only negligibly. An important observation is that the spectrum of modified heme undergoes no further change with peroxynitrite concentrations higher than 1.05 mM, which is consistent with a quantitative conversion of the heme. However, even 3.5 mM peroxynitrite concentrations did not affect the CaM bound to NOS-II, indicating that CaM does not become a target for peroxynitrite even after complete chemical modification of the heme. Considering the potential impact of ONOO-/ONOOH on the functional activity of CaM and the importance of CaM for the structure of NOS-II, we conclude that formation of the largely Ca2+-independent and irreversible complex between CaM and NOS-II is absolutely necessary for the continuous production of NO• in biological systems. However, though exposure of NOS-II to high concentrations of peroxynitrite did not yield oxidized CaM, even low fluxes of peroxynitrite abolished the activity of NOSII. NOS-II activity requires association of the protein monomers to give the catalytically active dimer (37, 38). Gel filtration analysis indicated that our preparations contained ∼50% of the dimer when the purification was conducted without H4B in the buffer. Addition of H4B to the preparation increased the level of the dimer to >80%. A precise correlation between NOS-II assembly and oxidant susceptibility was not possible, but formation of predominantly dimeric enzyme due to the addition of H4B did not protect the enzyme from inactivation by ONOO-/ONOOH. Relatively short exposure of the NOS-II preparations to low steady state concentrations of ONOO-/ONOOH inactivated the enzyme and inhibited NO• production. The decrease in the activity correlated with a change in the Soret region of the absorption spectrum, i.e., a decrease in the intensity and a shift of the λmax from 419 to 409 nm. Correlation of the absorbance maximum of NOS-II exposed to ONOO-/ONOOH with that of H2O2treated enzyme suggests that both reagents cause similar changes, possibly reflecting the initial formation of a ferryl intermediate, in the nature of the prosthetic heme group. The report that a ferryl iron species is formed when iron(III) porphyrins are exposed to ONOO-/ ONOOH is of relevance in this context (39). In contrast to the Fe(III) porphyrins, however, rearrangement of the ferryl iron intermediate to yield the Fe(III) porphyrin and inorganic nitrate was not observed in the case of NOSII. Interestingly, although H4B did not prevent the peroxynitrite-induced inactivation of NOS-II, it modulated the peroxynitrite-mediated spectral change, resulting in a shift of the Soret band to 420 nm rather than to 409 nm. A possible role for H4B as a protective element


was suggested by Griscavage et al. (40), but this apparently does not apply to peroxynitrite. H4B influenced the peroxynitrite-dependent modifications of the heme group or its environment but did not prevent the inactivation of NOS-II by ONOO-/ONOOH. Experiments are currently in progress to determine whether, in the presence of H4B, the NOS-II protein skeleton itself is chemically modified by peroxynitrite. The present data suggest that an increased and prolonged activity of NOS-II in cells can only be maintained in the absence of even low concentrations of peroxynitrite. Although most of the macrophage-derived peroxynitrite may actually exist outside the cell, it has been shown that peroxynitrite can cross cell membranes (11, 17). Thus, peroxynitrite may reach target molecules such as NOS-II inside macrophages. The nature of NOSII inactivation by peroxynitrite is unclear at present, but a contribution from CaM oxidation can be excluded due to inaccessibility of the sensitive CaM residues within the NOS-II/CaM structure.

Acknowledgment. This work was supported by a grant from the American Heart Association (KS-94-GB3) to C.S. and by the National Institutes of Health grants PO1AG12993-01 (subproject 1) to C.S. and GM25515 to P.R.O.M.

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TX960188T

Inactivation of the Inducible Nitric Oxide Synthase by Peroxynitrite

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