Structure of the Photoreactive Iron Center of the Nitrile Hydratase from Rhodococcus sp. N-771

Nitrile hydratase (NHase) fromRhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the α subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu-Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from αIle107 to αTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the α subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.

ing amides (1,2). NHase consists of two kinds of subunits (␣ and ␤ with the molecular mass values of 23 kDa) and contains non-heme iron (3) or non-corrinoid cobalt (4) atoms. The NHase from Rhodococcus sp. N-771, which is a ␣␤ heterodimer containing a non-heme iron, is inactivated by aerobic incubation in the dark for a half-day (dark-inactivation), whereas the enzyme purified from the dark-inactivated cells is immediately converted to the active form by light irradiation (photoactivation) (5,6). Recently, it has been revealed that dark-inactivation and photoactivation are controlled by association and photodissociation of nitric oxide (NO) with the non-heme iron center (7)(8)(9). Similar photosensitivity is observed in the NHases from Rhodococcus sp. N-774 (10) and R312. 2 These NHases seem to be identical to the one from Rhodococcus sp. N-771 because of the same nucleotide sequences (11,12). 3 The iron-containing NHase is the first enzyme with a mononuclear low-spin non-heme iron(III) which is thought to be involved in the catalysis (3). The structure of the iron center in the active form has been studied by various spectroscopies including ESR (3), resonance Raman (13), extended x-ray absorption fine structure (13) and electron nuclear double resonance (14), and the ligand-donor set of N 3 OS 2 has been proposed (14), which is supported by model complexes of the iron center (15)(16)(17). Recently, the metal site structure has been studied in detail by means of electron nuclear double resonance (18), resonance Raman (19), and x-ray absorption (20) spectroscopies. It was demonstrated that the coordination sphere of the metal site consisted of two cis-coordinated sulfhydryl ligands, three His imidazole ligands, and an exchangeable solvent ligand, probably an hydroxo group. It is widely believed that the metal site structure of the cobalt-containing NHases is closely related with that of the iron-containing one. Indeed, recent ESR and extended x-ray absorption fine structure studies showed that the metal site of the NHase from Rhodococcus rhodochrous J1 was a mononuclear low-spin six-coordinate Co 3ϩ ion with the ligand field of mixed sulfur, and nitrogen or oxygen donor atoms (4).
In previous reports, we found that the iron center bound with NO is solely located on the ␣ subunit (21,22). Interestingly, the ␣ subunit denatured by 6 M urea exhibited light-induced UV-Vis absorptional change similar to the native NHase, indicating that the nitrosylated iron center was stably associated with the ␣ subunit even unfolded (21). Moreover, Duine and * This work was supported in part by a grant from the "Biodesign Research Program" from the Institute of Physical and Chemical Research (RIKEN) and by a Grant-in-Aid for Scientific Research on Priority Areas "Molecular Biometallics" (0824946) from the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  colleagues 4 digested the inactive NHase with Pronase, and obtained a peptide containing chromophore with an absorption peak characteristic of the inactive form. These findings suggest that the iron atom binds with a short specific region of the ␣ subunit. In the present study, we analyzed the iron-binding site through proteolytic cleavage of the ␣ subunit followed by reversed-phase HPLC, and found that the nitrosylated iron center complex consists of a segment of 11 amino acids of the ␣ subunit, which contains the highly conserved Cys cluster. Furthermore, we have shown by mass spectrometry, protein sequence, and amino acid composition analyses that a Cys residue in this region is post-translationally oxidized to a cysteinesulfinic acid (Cys-SO 2 H). Based on the results, we will discuss the structure of the photoreactive non-heme iron center of the NHase in the inactive form.

EXPERIMENTAL PROCEDURES
Preparation of the Inactive and Active Forms of the NHase and the ␣ Subunits-Dark-inactivated NHase was prepared as described (21). For photoactivation, the NHase in the inactive form was irradiated with a photoreflector lamp (500 W SPOT, Toshiba) for 15 min in an ice bath. The ␣ subunit was isolated from the inactive form by anion-exchange chromatography in the presence of 6 M urea in the dark, while the isolation from the active form was carried out under light (21). The ␣ subunit isolated from the inactive form reconstituted the NHase with the ␤ subunit. The reconstituted complexes exhibited about 80% of the specific activity of the native NHase after photoactivation. However, the ␣ subunit from the active form did not reconstitute the NHase at all (21). The inactive enzyme and the isolated ␣ subunits were stored as suspensions in 60% saturated ammonium sulfate at 4°C in the dark.
Preparation of the Recombinant ␣ Subunit-The NHase operon of Rhodococcus sp. N-771 has been cloned and sequenced. 3 The N-terminal half of the ␣ subunit gene was amplified by polymerase chain reaction to introduce an NdeI site at the initiation codon. The fragment was digested by NdeI and PstI, whose digest site is located just upstream of the complementary polymerase chain reaction-primer binding site, and subcloned into pET-3c with the remainder of the ␣ subunit gene. Escherichia coli, BL21(DE3), was transfected with the constructed expression vector, pRCM␣, and the recombinant ␣ subunit (␣ rec ) was expressed as inclusion bodies. Cells were resuspended in 50 mM Tris-HCl, pH 7.5, and disrupted by sonication. After removal of the supernatant by centrifugation (40,000 ϫ g, 20 min), the precipitate was washed with 1 M sucrose and then 2% Triton X-100 and 10 mM EDTA. The washed precipitate was solubilized with 50 mM Tris-HCl, pH 7.5, containing 6 M urea and 2 mM 2-mercaptoethanol and applied to an anion-exchange Q-Sepharose column (Pharmacia, 2.5 cm inner diameter ϫ 10 cm) equilibrated with the same buffer. ␣ rec was eluted with a 0 -500 mM NaCl linear gradient at a flow rate of 1.5 ml/min. ␣ rec was estimated to be more than 99% pure by SDS-polyacrylamide gel electrophoresis, and stored at 4°C as a suspension in 60% saturated ammonium sulfate.
To minimize the size of the iron center complex peptide, the tryptic peptide was further digested. The peptide (1.35 g) in 20 mM Tris-HCl, pH 7.5, was treated with 1 g of thermolysin for 1 h at 37°C, and then with carboxypeptidase Y (1 g) and leucine aminopeptidase M (1 g) for 12 h at 37°C.
Preparation of the Peptide Fragments from the Active NHase-The ␣ subunit isolated from the active enzyme was cleaved with trypsin without being shielded from light. The digest was separated by reversed-phase HPLC with an Aquapore RP300 column (4.6 ϫ 100 mm, Perkin-Elmer Applied Biosystems). Solvent A was 0.09% trifluoroacetic acid, and solvent B was 0.075% trifluoroacetic acid containing 80% acetonitrile. The column was equilibrated with 100% solvent A and eluted with a linear gradient from 0 to 80% solvent B over a period of 16 min at a flow rate of 0.5 ml/min.
Mass Spectrometry Measurements-The molecular masses of proteins and peptide fragments were determined by fast atom bombardment-mass spectrometry (FAB-MS) or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). FAB-MS spectra were obtained with a JEOL JMS-HX110 mass spectrometer operated at 10-kV accelerating voltage and a resolution of 1:3000. Ionization was carried out using xenon gas as the primary beam with the energy of 6-keV in the positive mode. Samples were mixed with 0.1% trichloroacetic acid in 3-nitrobenzyl alcohol/thioglycerol/glycerol (1:1:1). MALDI-TOF MS was performed on a Reflex (Bruker) equipped with a delayed extraction ion source. Samples were prepared by mixing 0.5 l of the 10 mg/ml 2,5-dihydroxybenzoic acid matrix solution with 0.5 l of the sample dissolved in 33% acetonitrile, 0.07% trifluoroacetic acid on the target. Positive ion mass spectra were acquired in reflectron mode at 28.5-kV accelerating voltage. Mass scale was calibrated using commercially available peptides.
Identification of the Modification of ␣Cys 112 -An aliquot of the irradiated ␣ subunit (800 g) was carboxymethylated after reduction, and then treated with trypsin (5 g). The carboxymethylated NK24 was isolated by reversed-phase HPLC as described above. After removal of acetonitrile in a vacuum centrifuge and adjustment of the pH value to 8.0, the carboxymethylated NK24 was cleaved with thermolysin (1.0 g) at 37°C for 6 h, and then analyzed by reversed-phase HPLC with a Superpher 60RP select B column (2.1 ϫ 125 mm, Merck). Solvent A was 0.09% trifluoroacetic acid, and Solvent B was 0.075% trifluoroacetic acid containing 80% acetonitrile. The column was equilibrated with 20% solvent B and eluted with a linear gradient from 20 to 55% solvent B over a period of 17.5 min at a flow rate of 0.2 ml/min. After removal of acetonitrile in a vacuum centrifuge and adjustment of the pH to 8.0, the fraction containing an octapeptide, LP8 (␣Leu 111 to ␣Pro 118 ), was incubated with leucine aminopeptidase M (0.02 g) for 6 h at 37°C. Once dried in a vacuum centrifuge, the digest was dissolved in 5 l of acetonitrile and incubated with 5 l of 18 mM (ϩ)-1-(9-fluorenyl)ethyl chloroformate (FLEC) (23) in acetone for 10 min at room temperature. The reaction was terminated by addition of 10 l of 100 mM tryptophan in 100 mM borate buffer at pH 9.0. The sample solution was mixed with 10 l of 9 N acetic acid and 40 l of 100 mM sodium acetate buffer, pH 4.0, and then subjected to reversed-phase HPLC with a PEGASIL ODS-3 column (4.6 ϫ 250 mm, Senshu) equilibrated with 0.1 M sodium acetate (NaOAc) buffer, pH 4.0, NaOAc/acetonitrile/tetrahydrofuran (77/15/8, v/v). The column was isocratically run with the same buffer at a flow rate of 0.6 ml/min.
Other Procedures-The concentration of the NHase was estimated by the absorbance of the inactive form at 280 nm (⑀ 280 ϭ 1.7 ml⅐mg Ϫ1 ⅐cm Ϫ1 ) (21). The concentrations of the isolated subunits were determined with Coomassie Brilliant Blue dye reagent (24) using the inactive NHase as a standard. Carboxymethylation was carried out by the method of Crestfield et al. (25). Before carboxymethylation, the sample was reduced by incubation in 1 M Tris-HCl, pH 8.5, containing 10 mM EDTA and the 100-fold excess of dithiothreitol at 37°C for 1 h. ESR spectra were measured with a JEOL JES FE-3AX X-band ESR spectrometer. Iron atoms were quantified using an inductively coupled plasma mass spectrometer (VG Elemental, VG PQ⍀). N-terminal sequence and amino acid composition were analyzed with a 477A protein sequencer connected on line to a 120A PTH analyzer (Perkin-Elmer Applied Biosystems) and an 835 Amino Acid Analyzer (Hitachi), respectively.
Materials-TPCK-treated trypsin (type, XIII: from bovine pancreas), carboxypeptidase Y, leucine aminopeptidase M, L-cysteine-sulfinic acid, and DL-cysteic acid were purchased from Sigma, and thermolysin from Seikagaku Kogyo (Tokyo, Japan). N-Methyl-D-glucamine dithiocarbamate (MGD) was a generous gift from Dr. I. Ueno of RIKEN. 3-Nitrobenzyl alcohol, thioglycerol, and glycerol were the products of Tokyo Kasei (Tokyo, Japan), and 2,5-dihydroxybenzoic acid was purchased from Aldrich. All the other reagents used were of the highest biochemical grade available.

RESULTS
Proteolysis of the ␣ Subunit of Inactive NHase-We first cleaved the ␣ subunit with trypsin in the dark. Even after cleavage, the absorption peak at 370 nm, characteristic of the inactive form, remained and disappeared upon light irradiation (data not shown). The excitation profile of the resonance Raman spectra of the inactive form suggested that this band was due to the transitions with both the iron 4 NO and iron 4 S charge transfer characters (9). To obtain the photoresponsive peptide, the digest was separated by reversed-phase HPLC on a column connected to a diode array detector. Since the absorbance at 370 nm readily disappeared both below pH 6 and above pH 9, the pH values of the solvents used in HPLC were controlled at 7.5. The chromatogram at 215 nm showed 13 peaks while that at 370 nm contained only one peak at 20.6 min (Fig.  1, A and B). Protein sequence analysis revealed that the 20.6min peak contained a peptide fragment of 24-amino acids, Asn 105 -Val-Ile-Val-(Cys)-Ser-Leu-(Cys)-Ser-(Cys)-Thr-Ala-Trp-Pro-Ile-Leu-Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys 128 ((Cys) was assumed as a Cys residue based on the nucleotide sequence) (NK24). This region is highly conserved among all known NHases and contains the Cys cluster (Cys 109 -Ser-Leu-Cys 112 -Ser-Cys 114 ). When the tryptic digest was irradiated prior to injection, the elution profile monitored at 215 nm was identical to that of the non-irradiated digest except that the 20.6-min peak delayed to 22.4 min (Fig. 1C). Since the amino acid sequences were identical, the shift would be induced by release of NO and/or iron. Fig. 2 shows the UV-Vis difference spectrum between the 22.4-min peak of the irradiated digest and the 20.6-min peak of the non-irradiated one. It exhibited two positive peaks at 280 and 370 nm, which is in good agreement with the photo-induced absorptional change of the isolated ␣ subunit (21).
Detection of Iron Atoms and NO in NK24 -We investigated the quantitative measurements of both the iron atom and NO in NK24 isolated from the inactive enzyme. The amount of iron atoms in NK24 was measured to be 0. was also ESR silent (Fig. 3B). However, when [(MGD) 2 -Fe II ] was added to the solution in the dark, clear ESR signals of [(MGD) 2 -Fe II -NO] (a N ϭ 1.29 millitesla and g iso ϭ 2.04) were observed (Fig. 3C), demonstrating that this peptide intrinsically possessed NO molecules. Unlike the native NHase in the inactive form, the ESR spectrum appeared without light stimulation. The reason for this discrepancy is unknown. However, it is probable that [(MGD) 2 -Fe II ] attacks the nitrosyl iron in NK24 because of the absence of other protein moieties.
Isolation of the Nitrosylated Iron Center Complex-To obtain the minimum peptide segment required for the nitrosylated iron center, we attempted to digest NK24 with amino-and carboxypeptidases in the dark. At first, NK24 (450 pmol) was treated with thermolysin at a molar ratio of 1:10 so that it could be efficiently digested by exopeptidases. Subsequently, carboxypeptidase Y and leucine aminopeptidase M were added at a molar ratio of 1:50, and the reaction mixture was incubated for 12 h at 37°C. The digest was analyzed by reversed-phase HPLC in pH 7.5 in the dark (Fig. 4). A peak at 11.6 min contained a peptide of 11 amino acids from Ile 107 to Trp 117 (IW11) while other peaks contained hydrolyzed amino acids and buffer components. The yield of IW11 was estimated to be 28%. The UV-Vis absorption spectrum of the 11.6-min peak exhibited the absorption peak at 370 nm (inset of Fig. 4), indicating that the nitrosyl iron was still associated with IW11. When NK24 was irradiated prior to digestion with thermolysin and exopeptidases, the 11.6-min peak completely disappeared (data not shown). Photodissociation of NO might destabilize the iron center, and induce the subsequent proteolysis of the peptide. The spectra of the 20.6-min peak of the non-irradiated digest (Fig. 1A) and of the 22.4-min peak of the irradiated one (Fig. 1C) were obtained with a diode array detector in Hewlett-Packard HP1090M liquid chromatograph. The difference spectrum was obtained by subtracting the latter from the former.  Fig. 5 shows FAB-MS spectrum of NK24 prepared from the photoactivated NHase (NK24 active ). The iron atom was dissociated from NK24 by reversed-phase HPLC in the presence of trifluoroacetic acid. The molecular mass of NK24 active was m/z 2693.6, which is 30.3 Da larger than that deduced from the corresponding gene sequence. The molecular mass of the isolated ␣ subunit was also about 30 Da larger than the expected value by electrospray (data not shown). These cannot be attributed to nitrosothiolation of side chains of Cys residues by NO released from the iron center, because NO is quantitatively released into the solvent upon photoactivation (8) and the nitrosothiol group is unstable in the acidic solvent used in reversed-phase HPLC for isolation of the peptide. Thus, it was suggested that the NHase have a post-translational modification in this region of the ␣ subunit.

Post-translational Modification of the Cys Residue in NK24 -
Sequence analysis of NK24 active indicated that all residues were normal except three Cys residues, which are not stable in automated Edman degradation without alkylation of the sulfhydryl groups. Therefore, it was most probable that one or two Cys residues were modified to cause a mass increase of 30 Da. To identify the modified Cys residue(s), we carboxymethylated the Cys residues after reduction. Cys 109 and Cys 114 were identified as carboxymethylcysteines, whereas Cys 112 remained undetectable. The molecular mass of reduced and carboxymethylated NK24 active was determined by MALDI-TOF MS to be 2810.9 (Fig. 6A), which is 31.6 Da larger than the expected value (the molecular mass calculated from the gene sequence (2663.3) plus that of two carboxymethyl groups (ϩ59 ϫ 2 Ϫ H ϫ 2)). Thus, the 112th Cys residue of the ␣ subunit must be specifically modified to cause the mass increase of 32 Da.
To rule out the possibility that the modification is an artifact caused during the preparation of NK24, we analyzed the corresponding fragment prepared from the recombinant ␣ subunit (␣ rec ) expressed in E. coli. Like the native ␣ subunit, ␣ rec was purified by anion-exchange chromatography in the presence of 6 M urea. The purified ␣ rec was colorless, indicating the absence of an iron center (data not shown). This ␣ rec was readily cleaved with trypsin and carboxymethylated after reduction. The cor-responding fragment was purified by reversed-phase HPLC and analyzed by sequence analysis and MALDI-TOF MS (Fig.  6B). All three Cys residues in the fragment were carboxymethylated and the molecular mass of the fragment was 2837.0 Da, which well coincided with the expected value, 2837.3 Da (the molecular mass calculated from the gene sequence (2663.3) plus that of three carboxymethyl groups (ϩ59 ϫ 3 Ϫ H ϫ 3)). Therefore, the modification of the 112th Cys residue seems to be specific to Rhodococcus sp. N-771.
Identification of the Post-translational Modification of the 112th Cys Residue-Finally, we studied the structure of the modified Cys 112 . The molecular mass increment of 32 Da can likely be explained by derivatization of the 112th Cys residue by one sulfur atom (Cys-S-SH) or two oxygen atoms (cysteinesulfinic acid (Cys-SO 2 H); Fig. 7). Since Cys-S-SH should be carboxymethylated after reduction, it was assumed that Cys 112 was modified to Cys-SO 2 H. We subjected the peptide to amino acid analysis through derivatization with FLEC (23). Avoiding oxidation during hydrolysis in acidic conditions, NK24 active was . The elution conditions were as follows. Solvent A was 20 mM ammonium acetate, pH 7.5, and solvent B was 20% solvent A ϩ 80% acetonitrile. The column was equilibrated with 100% solvent A and eluted with a linear gradient from 0 to 80% solvent B over a period of 16 min at a flow rate of 0.2 ml/min. The inset shows the UV-Vis absorption spectrum of the elution at 11.6 min. The spectrum was obtained by the same diode array detector as Fig. 1. hydrolyzed enzymatically. We prepared NK24 active from the irradiated ␣ subunit whose sulfhydryl groups were blocked by carboxymethylation after reduction in advance. Since Cys 112 is the eighth amino acid residue from the N terminus of the peptide, we further cleaved the peptide with thermolysin prior to exopeptidase hydrolysis. The resulting octapeptide, LP8 active (Leu 111 -X-Ser-*Cys-Thr-Ala-Trp-Pro 118 ; *Cys designates carboxymethylcysteine), was purified by reversed-phase HPLC. The molecular mass of the octapeptide estimated by MALDI-TOF MS was m/z 970.7, 32.6 Da larger than that calculated from the gene sequence. The chromatograms of amino acid analysis of the authentic DL-Cys-SO 3 H and L-Cys-SO 2 H after FLEC treatment are shown in Fig. 8, A and B. FLEC derivatives of L-Cys-SO 3 H, D-Cys-SO 3 H, and L-Cys-SO 2 H (FLEC-L-Cys-SO 3 H, FLEC-D-Cys-SO 3 H, and FLEC-L-Cys-SO 2 H) were eluted at 22.9, 23.4, and 26.7 min, respectively. About 40% of the authentic L-Cys-SO 2 H was oxidized to L-Cys-SO 3 H. When LP8 active hydrolyzed with leucine aminopeptidase M was subjected to amino acid analysis after FLEC treatment, the peaks of FLEC-L-Cys-SO 2 H as well as FLEC-L-Cys-SO 3 H were observed (Fig. 8C). FLEC derivatives of other amino acids were eluted after 43 min (data not shown). The amount of FLEC-L-Cys-SO 2 H and FLEC-L-Cys-SO 3 H was 44 and 55 pmol, respectively. The yield of the Leu residue at the N terminus was estimated to be 97.3 pmol, thus the octapeptide contained one L-Cys-SO 2 H per molecule. From these results, we concluded that the 112th Cys residue of the ␣ subunit is post-translationally oxidized to a sulfinic acid derivative in the intact NHase from Rhodococcus sp. N-771.

Cysteine-Sulfinic Acid Stabilization in the Iron Center of
NHase-Mass spectrometry is a useful technique for identification of a post-translational modification in a protein (27). Using this technique, we have shown that Cys 112 stably exists as a sulfinic acid derivative in the Rhodococcus N-771 NHase. It is unlikely that the derivatization is an artifact because Cys 112 is specifically oxidized among the Cys-cluster (Fig. 6A) and because the recombinant ␣ subunit expressed in E. coli (␣ rec ) contains no derived Cys residues despite almost the same purification procedures (Fig. 6B). It is unknown how ␣Cys 112 is post-translationally modified to Cys-SO 2 H. NO molecules might oxidize ␣Cys 112 in Rhodococcus sp. N-771. There are several reports describing that NO and ONOO Ϫ , which is produced from O 2 Ϫ and NO, indeed oxidize protein sulfhydryls (28,29). Unfortunately, ␣ rec does not reconstitute the NHase with the ␤ subunit (data not shown) and, therefore, we cannot investigate whether Cys-SO 2 H is essential for the photoactivation and/or the catalysis. The inability to reconstitute the NHase is not due solely to the absence of the Cys-SO 2 H derivatization, because the ␣ subunit isolated from the active form also does not reassemble with the ␤ subunit (21). The sulfinic acid derivative is stabilized in the native inactive NHase and does not undergo further oxidation even under aerobic conditions as long as it is kept in the dark, strongly suggesting that the Cys 112 residue coordinates to the iron atom. The coordinated atom is the sulfur atom or most likely one of the oxygen atoms in the sulfinic acid group because the donation effect of the sulfur atom must be significantly weakened by two bound oxygen atoms.
Another example of a protein containing Cys-SO 2 H is SP-22, an unidentified substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex (30). SP-22 is a homoligomer consisting of M r 21,600 subunits, and the 47th amino acid of which was found to be a sulfinic acid by FABmass spectrometry. The fact that the vicinal sequence is highly conserved among several homologous proteins suggests that this residue is important to the biological function of SP-22. It remains possible that this residue exists as a cysteine-sulfenic acid (Cys-SOH) or a normal cysteine in the native SP-22 protein. On the other hand, it has been shown that two flavoproteins, NADH oxidase and NADH peroxidase from Enterococcus faecalis 10C1, possess Cys-SOH residues at non-flavin redox centers in the native enzymes (31,32). These modified Cys residues play an essential role in the catalysis, being switched between Cys-SOH and Cys-S Ϫ during turnover (31). Furthermore, Cys-SOH/Cys-SH redox cycle may be important for DNA binding activity of transcription factors including E2 protein (33), OxyR (34,35), Jun and Fos (36), and nuclear factor I (37). However, none of these proteins are metalloproteins and the amino acid sequences around the modified Cys residues show no homology with the NHase. Therefore, the role of Cys-SO 2 H in NHase may be unique.
Structure of the Iron Center of the NHase in the Inactive Form-Cys 109 and Cys 114 of NK24 prepared from the inactive form were not modified by carboxymethylation even after reduction (data not shown), whereas those from the active form were carboxymethylated by the same treatment (Fig. 6A). Thus, the sulfhydryl groups of these Cys residues are stabilized by coordination to the nitrosyl-iron. The results of the present study suggested that, in the inactive state, the ligands in the six-coordinate sphere of the non-heme iron center include two cysteine thiolates, and one oxygen atom of sulfinic acid and one NO molecule. A model of the non-heme iron center of the NHase in the inactive form is illustrated in Fig. 9. The other two ligands remain unknown. Previous spectroscopic studies of the active enzyme suggested the coordination of His imidazole ligands at these sites (14,20). However, very recently the crystal structure of the Rhodococcus R312 NHase in the active form was unveiled and it was revealed that, in the active form, the ligands to the iron are the side chains of ␣Cys 110 , ␣Cys 113 , and ␣Cys 115 (corresponding to ␣Cys 109 , ␣Cys 112 , and ␣Cys 114 of the Rhodococcus N-771 enzyme), 5 and the main chain amide nitrogens of ␣Ser 114 and ␣Cys 115 (corresponding to ␣Ser 113 and ␣Cys 114 of the Rhodococcus N-771 enzyme) 5 (38). Consistently with the crystal structure, the fact that IW11, a minimum peptide segment, does not contain a His residue as well as potential side chains nitrogen donors such as Asn, Gln, and Arg, suggests the coordination of backbone amide nitrogen atoms in IW11.

CONCLUDING REMARKS
We have isolated the peptide complex containing the nitrosylated non-heme iron center from the ␣ subunit of the inactive NHase. The minimum peptide complex obtained (IW11) consisted of the peptide from ␣Ile 107 to ␣Trp 117 , an iron atom, and NO molecules. Although the possibility of ligand exchange during the isolation procedure of the peptide complex cannot be ruled out, the fact that all the ligand amino acids of the active form are included in IW11 suggests that the ligand amino acid residues of the iron center is basically conserved between the inactive and active form of the NHase. In other words, photoactivation is probably induced by the replacement of NO with a solvent ligand in the iron center.
␣Cys 112 is post-translationally modified to a cysteine-sulfinic acid (Cys-SO 2 H) in the native NHase. The biological role of Cys-SO 2 H in the photoresponse and/or catalysis is still unclear but, so far as we know, this is the first report on the presence of a Cys-SO 2 H in the metalloprotein.
Two sulfur atoms of ␣Cys 109 and ␣Cys 114 , and one oxygen atom of ␣Cys-SO 2 H-112 are probably coordinated to the nitrosyl iron atom in the inactive form. Our model is contradictory to the metal site structure of the active form with respect to the modification of ␣Cys 112 to sulfinic acid in the Rhodococcus N-771 enzyme. The reason for this discrepancy remains unclear. The structure of the Rhodococcus N-771 enzyme may be different from that of the Rhodococcus R312 enzyme. X-ray crystallographic analysis of the inactive form of the Rhodococcus N-771 enzyme is currently underway in our laboratory (39). There is no data to suggest the iron center bonds with an oxygen atom of Cys-SO 2 H except for the results of this study. Model complexes having similar iron coordination spheres should be investigated. Also, it should be clarified whether the corresponding Cys residues of other iron-and cobalt-containing NHases are modified by sulfinic acid derivatives. FIG. 9. Model of the non-heme iron center in the inactive form of the NHase. S1,2 are sulfur atoms of Cys 109 and Cys 114 ; O is the oxygen atoms from the 112th cysteine-sulfinic acid; and X and Y are unidentified.