©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Nitric Oxide Synthase as a Thiolate-ligated Heme Protein Using Magnetic Circular Dichroism Spectroscopy
COMPARISON WITH CYTOCHROME P-450-CAM AND CHLOROPEROXIDASE (*)

(Received for publication, April 7, 1995; and in revised form, June 16, 1995)

Masanori Sono (1) Dennis J. Stuehr (3) (4) Masao Ikeda-Saito (4) John H. Dawson (1) (2)

From the  (1)Department of Chemistry and Biochemistry and the (2)School of Medicine, University of South Carolina, Columbia, South Carolina 29208, the (3)Department of Immunology, the Cleveland Clinic, Cleveland, Ohio 44195, and the (4)Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nitric oxide (NO) has recently been recognized as an important biomolecule playing diverse physiological roles. It is synthesized in several different tissues from L-Arg and O(2), using NADPH as an electron donor, by a family of heme-containing catalytically self-sufficient monooxygenases known as nitric oxide synthases (NOS). Recently, the CO complex of reduced NOS has been shown to exhibit an absorption maximum near 450 nm, a characteristic spectral feature of cytochrome P-450 (P-450). Yet, the amino acid sequences of NOS and P-450 have no homology. To further probe the active site heme coordination structure and the heme environment of NOS, we have employed magnetic circular dichroism (MCD) and CD spectroscopy in the present study. MCD spectra of several derivatives of rat brain neuronal NOS strikingly resemble those of analogous derivatives of bacterial P-450-CAM and fungal chloroperoxidase, two known thiolate-ligated heme proteins. Given the proven fingerprinting capability of MCD spectroscopy, this provides convincing evidence for endogenous thiolate (cysteinate) ligation to the heme iron of NOS. Furthermore, the heme-related Soret CD bands of NOS (positive) and P-450s (negative), as represented by P-450-CAM, are almost mirror images, whereas chloroperoxidase exhibits totally different CD band shapes. This suggests that the active sites of NOS and P-450 may share some common structural features, but significant distinctions exist between their heme environments in certain aspects such as hydrophobicity or size.


INTRODUCTION

Nitric oxide (NO), (^1)a moderately reactive (relatively unstable) and potentially toxic inorganic free radical gas, has recently been recognized as an important biomolecule playing diverse physiological roles. It is synthesized in the brain cerebulleum as a neurotransmitter, in vascular endothelial cells as an ``endothelial-derived relaxing factor'' which helps control blood pressure, and in macrophages as a cytotoxic agent(1, 2, 3, 4, 5, 6) . The biosynthesis of NO in these tissues is carried out from L-Arg and O(2) by a family of enzymes called nitric oxide synthases (NOS) (130-160 kDa) which utilize NADPH as an electron donor. The overall reaction is a two-step oxidative conversion of L-Arg to NO and L-citrulline via N^G-hydroxy-L-Arg as the intermediate(7) . The brain neuronal and macrophage NOSs are soluble while endothelial NOS is membrane bound with its N-terminal myristoylated (i.e. attached with n-tetradecanoic acid)(1, 5, 6) .

NOS is unusual among oxygenases in that it employs all of the following five prosthetic groups, NADPH, FAD, FMN, iron protoporphyrin IX (heme), and H(4)biopterin(1, 3, 4, 5, 6) . The first four components serve as redox co-factors, but H(4)biopterin has been reported to regulate the enzyme activity as a structural stabilizer(1, 5, 6) . The binding sites for the flavins and NADPH co-factors are located in the C-terminal half (the reductase domain) and the binding site for heme in the N-terminal half (the oxygenase domain). Thus, NOS is a catalytically self-sufficient oxygenase and, in that respect, is similar to a recently discovered fatty acid monooxygenase from Bacillus megaterium, cytochrome P-450(8) , except for the H(4)biopterin requirement. Unlike P-450, however, NOS possesses an additional binding site for calmodulin, the Ca complex of which is absolutely required for the catalytic activity of NOS. It has been reported that NOS is only catalytically active in its homodimer form (1, 4) . The amino acid sequence of the reductase domain of NOS has been found to be strikingly similar to that of mammalian P-450 reductase (6) , which also has a high sequence homology to the reductase domain of P-450(8) . However, the heme-binding domain of NOS does not share significant amino acid sequence with P-450 monooxygenases (6) .

It has recently been reported that the CO complex of reduced NOS exhibits an absorption maximum at 443-447 nm(9, 10, 11) , a signature spectral feature of the P-450 enzymes. Recent resonance Raman spectral data of NOS are also consistent with thiolate ligation to the heme iron (12, 13) . These findings have added further interest in understanding the active site (i.e. the heme-binding site) structure of NOS in relation to its mechanism of action. The first step of the NOS-catalyzed conversion of L-Arg to NO and L-citrulline is N-hydroxylation (Scheme 1 in (10) ), which is one of the typical mono-oxygenation reactions carried out by P-450 enzymes(14, 15, 16) . However, the second step appears to proceed by a quite unusual mechanism(1, 4, 5, 17) . Therefore, it is important to further define the active site structure of NOS in close comparison with that of P-450. In view of this point, we have employed magnetic circular dichroism (MCD) as well as CD spectroscopy in this study to probe the active site structure of NOS using purified rat brain enzyme. MCD spectroscopy has been proven to have a powerful fingerprinting capability to characterize structurally undefined heme centers by spectral comparison to data for structurally defined iron porphyrins, both from synthetic model complexes and heme protein derivatives(18) . CD spectroscopy, on the other hand, can provide information about the environment surrounding the heme moiety since the method is sensitive to electronic interactions between the heme prosthetic group and nearby aromatic amino acid residues or peptide backbones(19) . We have found that MCD spectral features of all of the several NOS derivatives examined in this study strikingly resemble those exhibited by the analogous derivatives of two known thiolate-ligated heme proteins, P-450-CAM (used as a representative of P-450 monooxygenases) and chloroperoxidase(20) . This provides convincing support for an endogenous thiolate ligation to the heme iron of NOS. On the other hand, the major Soret CD bands of analogous derivatives of NOS and cytochrome P-450-CAM are almost mirror images, suggesting that their active site heme environments differ in certain aspects such as hydrophobicity or size.


EXPERIMENTAL PROCEDURES

Materials

CO and NO gasses were obtained from Matheson Co. All other chemicals were purchased from Sigma or Aldrich and used as received. Chloroperoxidase, purified from the fungus Caldariomyces fumago to geq95% homogeneity (A/A geq 1.43 at 4 °C and pH 6)(21) , was the kind gift of Prof. Lowell P. Hager, University of Illinois at Champaign.

Cell Culture, NOS Purification, and NOS Activity Assay

The rat brain neuronal NOS was purified to 90-95% homogeneity from human kidney cells expressing the recombinant enzyme using the two-column procedure (2`, 5`-ADP-Sepharose and Mono-Q columns) as described previously(10) . NOS fractions from the second column, which were eluted with 20 mM Bis-Tris buffer, pH 7.4, containing 3 mM dithiothreitol, 10% glycerol, 2 µM H(4)biopterin, 150 mM NaCl, and no L-Arg, were concentrated to 30 µM (in heme) in a Centricon 30 microconcentrator at 4 °C and stored at -70 °C until use. The enzyme activity was measured as described previously (10) .

Preparation of MCD/CD Samples

A solution of purified and concentrated ferric NOS without added L-Arg as described above was used for a series of MCD/CD measurements for different derivatives in the following order. After examinations of ``resting'' NOS (without added L-Arg), L-Arg (1 mM) was added to form the L-Arg-bound ferric enzyme. This species was then reduced with solid sodium dithionite under N(2) (deoxy-ferrous enzyme), followed by gentle bubbling with CO (ferrous-CO enzyme). In the absence of Cabulletcalmodulin complex, the reduction of the heme iron of NOS was slow and took over 30 min at 4 °C for completion. The reduction was monitored by the disappearance of the absorption peak of ferric NOS at 640 nm as well as by an overall spectral change. To generate ferrous-NO NOS, NO gas was anaerobically introduced into the ferrous-CO enzyme-containing cuvette. The heme-bound CO was slowly replaced with NO (in 30 min) as judged by a clear spectral change of the enzyme accompanying a single set of isosbestic points (432.5, 458, 517, and 558 nm). Complete formation of L-Arg-bound ferrous-NO NOS in this manner was confirmed in separate experiments where it was generated in the absence of CO. Ferric-NO NOS was prepared by introducing NO gas into a ferric NOS solution under anaerobic conditions in the presence of L-Arg in the same buffer as used for the other NOS derivatives. Protein (heme) concentrations were determined by the pyridine hemochromogen assay using an extinction coefficient of 34 mM cm for the alpha-peak (556 nm)(22) .

Optical Absorption and MCD/CD Spectroscopy

Optical absorption spectra were recorded with a Varian/Cary 210 or 219 spectrophotometer interfaced to IBM PCs. MCD/CD spectra were recorded with a Jasco J-500A spectropolarimeter equipped with a Jasco MCD-1B electromagnet operated at 1.41T. The J-500A was interfaced to an IBM PS/2 model 50 computer by a Jasco IF-500-2 interface unit. Data acquisition and handling were performed as described elsewhere(23) . All spectral measurements for the NOS samples were carried out at 4 °C using a 0.1 cm (or 1 cm only for pyridine hemochromogen measurements) quartz cuvette attached with a glass joint at its top, which was sealed with a rubber septum for anaerobic experiments. Complete formation (reduction or ligand binding) of each enzyme derivative and the lack of enzyme denaturation were checked by recording optical absorption spectra of the samples before and after each MCD/CD scan; no more than 5% changes were considered acceptable.


RESULTS AND DISCUSSION

Extinction Coefficients for NOS

Since the previously reported millimolar extinction coefficients () for the brain and macrophage NOS isozymes in their native ferric (resting) and ferrous-CO derivatives (10) are considerably smaller (by 30-40%) than the typical values of P-450s(24, 25) , we have carefully redetermined the values for NOS by the pyridine hemochromogen method. The new values determined for substrate (L-Arg)-bound ferric NOS ( = 100 ± 3 mM cm) and ferrous-CO NOS ( = 121 mM cm) are indeed comparable to those of P-450-CAM(24) . These newly determined values for NOS are also in reasonable agreement with the values for chloroperoxidase (26) and for rat brain neuronal NOS reported previously by McMillan et al.(11) ( = 94 and = 124 mM cm, which are calculated from the absorption spectra displayed in Fig. 4A of (11) for resting NOS and its ferrous-CO derivative, respectively).


Figure 4: MCD (top) and optical absorption (UV-Vis) (bottom) spectra of the ferrous-NO derivative. L-Arg-bound NOS (-), camphor-bound P-450-CAM(- - -), and chloroperoxidase (bullet bullet bullet). The MCD and UV-Vis absorption spectra of P-450-CAM and chloroperoxidase are taken from (38) . The absorption spectrum of P-450-CAM is essentially identical to that reported by Peterson and co-workers(35) . The spectra for NOS below 370 nm (UV-Vis) and 330 nm (MCD) are omitted because of optical interference by a large amount of dithionite used. See the legend to Fig. 1and ``Experimental Procedures'' for the details for conditions and sample preparations for NOS.




Figure 1: MCD (top) and optical absorption (UV-Vis) (bottom) spectra of the five-coordinate ferric high spin derivative. Resting NOS without added L-Arg (--), L-Arg-bound NOS (-), camphor-bound P-450-CAM(- - -), and chloroperoxidase (bullet bullet bullet). The data for NOS were obtained at 4 °C with an enzyme concentration of 31 µM in 40 mM Bis-Tris buffer (pH 7.4) containing 3 mM dithiothreitol, 10% glycerol, 2 µM H(4)biopterin, and 150 mM NaCl with and without added 1 mML-Arg. The MCD spectra of P-450-CAM and chloroperoxidase are taken from Refs. 27 (P-450-CAM) and 20 (chloroperoxidase), and are essentially identical to those reported by Vickery et al.(28) and Dawson et al.(29) , respectively.



Optical Absorption and MCD Spectra of Ferric High Spin NOS and Its NO Complex

Overplotted in Fig. 1are optical (UV visible) absorption and MCD spectra of native ferric NOS with and without added L-Arg (1 mM), camphor-bound ferric P-450-CAM, and native ferric chloroperoxidase. The absorption spectra of the three heme proteins are more or less similar to one another except that NOS has extra absorption bands in the 440-520 nm region which are attributed to the flavin chromophores. Substrate-bound NOS exhibits a relatively broad Soret absorption peak at 393 nm and a MCD spectrum which is very similar to that of the ferric five-coordinate, high spin state of P-450-CAM (camphor-bound) (28) , chloroperoxidase(29) , and synthetic thiolate-ligated iron porphyrins (30) (spectra for the models not shown). A characteristic feature is an asymmetric MCD spectral band shape in the Soret region having a prominent trough centering at 395 nm which corresponds to the Soret absorption peak position for each of the three enzymes. The MCD trough intensity of NOS is similar to that of chloroperoxidase, but somewhat (by 40%) smaller than that of P-450-CAM. In the visible region between 440-700 nm, these three heme proteins also exhibit MCD spectral patterns very similar to each other and characteristic of a five-coordinate ferric high spin species(30) . As judged from the close MCD spectral similarity between L-Arg-bound NOS and P-450-CAM in the 440-500 nm region, the MCD signal from the NOS-bound flavin prosthetic groups appears to be insignificant.

Effects of addition of L-Arg (1 mM) to resting NOS on its optical absorption and MCD spectra were relatively small but clearly detectable as previously shown by difference absorption spectroscopy(31, 32) . The Soret absorption peak (396 nm) shifted to a shorter wavelength (393 nm) with a concomitant increase in its intensity by 10%. In the visible region, absorbance between 540-600 nm decreased slightly while the peak at 643 nm increased. As compared with substrate-bound NOS, the MCD spectrum of the resting enzyme has slightly smaller intensities at 395 (Soret trough), 425, and 560 nm, but increased intensities at 410, 520, and 580 nm. Based on the established spectral (both optical absorption and MCD) and spin-state changes accompanying the conversion of substrate-free ferric P-450-CAM (six-coordinate, low spin) to its camphor-bound form (five-coordinate, high spin)(20, 33, 34) , the observed spectral changes for NOS suggest that upon substrate binding, the predominantly high spin (mixed spin) state of ferric NOS becomes almost exclusively high spin.

NOS binds NO, its catalytic reaction product, to form a ferric-NO adduct (for its significance in catalysis, see the ferrous-NO NOS section described below) which exhibits absorption peaks at 442, 545, and 576 nm and a shoulder at 480 nm (spectrum not shown). The spectrum we obtained is very similar to that reported by Wang et al.(13) except for slight (4 nm) blue-shifts in the visible region peak positions for our sample and the lack of a shoulder between 600-650 nm. Ferric P-450-CAM (35) and chloroperoxidase (26) also form a complex with NO which exhibits sharp Soret and visible (alpha and beta) peaks. MCD spectral features of the NO adducts of the three heme proteins (not shown), which consist of three sets of derivative-shaped bands with crossover points corresponding to the absorption peak positions, are similar in band shapes, but distinguishable in band positions (red-shifted by 10-20 nm), from those observed for histidine-ligated heme proteins(36) .

Spectral Properties of Completely Substrate-free Ferric NOS

The optical absorption spectra of the purified resting (i.e. without added L-Arg) rat brain neuronal and macrophage NOS enzymes are indicative of a predominantly high spin state as judged from the presence of a charge-transfer band at 640 nm(10) . However, the Soret absorption peak position of resting NOS has been reported to occur from 397 to 406 nm(9, 10, 11) . In addition, the K value reported for the L-Arg complex of resting NOS also varies from 0.6 µM(32) to 2.5 µM(31) , although the kinetic K value is relatively constant between 2-3 µM(4) . This brings into question whether (a) these enzyme preparations were completely substrate-free and the resting NOS is inherently high spin like substrate-free P-450(25) , or (b) the enzyme preparations contained some tightly bound L-Arg which is responsible for the conversion of otherwise low spin resting NOS (like the majority of substrate-free P-450s) to a predominantly high spin species.

With regard to this point, high performance liquid chromatography analysis of L-Arg in purified resting NOS after denaturation indicated the presence of no more than 0.08 mol equivalent of L-Arg (per mol of heme) in the enzyme. (^2)Yet, predominantly high spin resting NOS could be converted to a low spin-type derivative (Soret peak at 416 nm with a shoulder at 390 nm) by gel-filtration column chromatography(32) , followed by concentration (0.5 mM), dilution (by 1000-fold), and storage overnight at 5 °C. (^3)The resulting sample was too low in concentration to be examined with MCD spectroscopy. Addition of L-Arg (1 mM) to this NOS sample regenerated a high spin species ((max) = 393 nm). These observations suggest that the predominantly high spin nature of resting NOS could result from an endogenous source within the protein which can be replaced with L-Arg or can slowly dissociate from the heme-binding site upon dilution of the enzyme. Thus, the true spectral nature of L-Arg-free native NOS and the identity of such an endogenous residue remain uncertain.

Optical Absorption and MCD Spectra of Deoxy-ferrous, Ferrous-CO, and Ferrous-NO Derivatives

Optical absorption and MCD spectra of dithionite-reduced NOS are overplotted with those of reduced P-450-CAM and chloroperoxidase in Fig. 2. Deoxy-ferrous NOS exhibits a Soret absorption peak at 412 nm, a noticeable shoulder at 450 nm, and a broad peak at 553 nm. These spectral features and band intensities of NOS are similar to those of deoxy-ferrous P-450-CAM and chloroperoxidase. The MCD spectra of the three heme proteins (Fig. 2) and of a five-coordinate thiolate-ligated ferrous heme model (spectrum not shown) (20, 29) are quite similar, especially in the Soret region where an asymmetric spectral pattern with a dominant trough at 420 nm is observed in all cases.


Figure 2: MCD (top) and optical absorption (UV-Vis) (bottom) spectra of the deoxy-ferrous (five-coordinate high spin) derivative. L-Arg-bound NOS (-), camphor-bound P-450-CAM (- - -), and chloroperoxidase (bullet bullet bullet). Note that the MCD spectra below and above 500 nm are plotted in different scales. The spectra below 360 nm for NOS and P-450-CAM are omitted because of optical interference by large absorbance ((max) = 315 nm) of dithionite used as a reductant. The MCD spectra of P-450-CAM and chloroperoxidase are taken from Refs. 27 (P-450-CAM) and 20 (chloroperoxidase), and are essentially identical to those reported by Vickery et al.(28) and Dawson et al.(29) , respectively. See the legend to Fig. 1and ``Experimental Procedures'' for the details for conditions and sample preparations for NOS.



Optical absorption and MCD spectra of substrate-bound ferrous-CO NOS are displayed in Fig. 3together with the previously reported spectra of P-450-CAM (37) and chloroperoxidase(26) . The three enzymes have absorption peaks in both the Soret and visible regions at nearly the same positions and exhibit MCD spectra similar to each other as well as to a thiolate-ligated ferrous-CO heme model (spectrum not shown)(20) . Their MCD spectra consist of a relatively intense, symmetrical derivative-shaped spectral features in the Soret region. In the visible region, the MCD spectrum of NOS is more similar to that of P-450-CAM than to that of chloroperoxidase which exhibits slightly better band shape resolution.


Figure 3: MCD (top) and optical absorption (UV-Vis) (bottom) spectra of the ferrous-CO derivative. L-Arg-bound NOS (-), camphor-bound P-450-CAM(- - -), and chloroperoxidase (bullet bullet bullet). Note that the MCD spectra below and above 480 nm are plotted in different scales. The spectra for NOS below 380 nm (UV-Vis) and 350 nm (MCD) are omitted because of optical interference by a large amount of dithionite used. The MCD spectra of P-450-CAM and chloroperoxidase are taken from Refs. 37 and 26, respectively. The MCD spectrum of P-450-CAM is similar to that reported by Vickery et al.(28) . See the legend to Fig. 1and ``Experimental Procedures'' for the details for conditions and sample preparations for NOS.



Substrate-bound ferrous-NO NOS exhibits optical absorption and MCD spectra which also closely resemble those of P-450-CAM and chloroperoxidase (38) as shown in Fig. 4. The optical absorption spectrum of the NOS complex is essentially identical to that reported by Wang et al.(13) . Ferrous-NO NOS has been shown to be formed as an inhibited species during the catalysis only under O(2)-limited conditions, although the ferric-NO derivative has not been detected under such conditions(13) . The MCD spectra of the three proteins in the Soret region are asymmetric and have a predominant peak at 430 nm. The overall features of the visible region MCD spectra of the three heme proteins are similar. However, the spectrum of ferrous-NO NOS is more similar to that of P-450-CAM than to that of chloroperoxidase which exhibits a slightly more resolved band pattern as in the ferrous-CO case.

CD Spectra of Ferric and Ferrous Derivatives

In Fig. 5, CD spectra of ferric (with and without added L-Arg) (A), deoxy-ferrous (B), ferrous-CO (C), and ferrous-NO (D) derivatives of NOS in the near UV and Soret regions (300-500 nm) are overplotted with the spectra of the analogous derivatives of P-450-CAM and chloroperoxidase. The CD spectra of all the four P-450-CAM derivatives (300-500 nm) (33, 37, 39) have previously been reported. At first glance one notices that the four derivatives of NOS exhibit positive major CD bands, which is in sharp contrast to the corresponding negative major CD bands for the P-450-CAM derivatives. As a result, the CD spectra of NOS and P-450-CAM are almost mirror images, especially for the high spin ferric form (Fig. 5A), although the CD band intensities (and the areas of CD bands) for the other derivatives of NOS are nearly 2-fold greater than those of P-450-CAM. The negative Soret CD band patterns of P-450-CAM are shared by other P-450s from many different sources, including liver microsomes(40) , adrenal cortex mitochondria(41) , and other bacteria, (^4)although the band intensity varies among different P-450s. Thus, P-450-CAM can be considered as representative of the other P-450s. The chloroperoxidase derivatives, on the other hand, exhibit more complex CD spectral patterns having both positive and negative bands with similar intensities.


Figure 5: Soret CD spectra of the ferric high spin (five-coordinate) (A), deoxy-ferrous (high spin, five-coordinate) (B), ferrous-CO (C), and ferrous-NO (D) derivatives. Resting NOS (--) (A only), L-Arg-bound NOS (-), camphor-bound P-450-CAM(- - -), and chloroperoxidase (bullet bullet bullet). The spectra of P-450-CAM are taken from Refs. 39 (A), 27 (B), and 37 (C and D) and are essentially identical to those (for A-C) reported by Peterson(33) , except that the intensities in (33) should be multiplied by 10^2. The CD unit used in this paper, Delta (the molar extinction coefficient, in mM cm) is related to [] (the molar ellipticity, in degbulletcm^2/decimole) as: Delta = []/2.303(4500/) 3.0 10 [] ((19) ). All of the chloroperoxidase spectra were obtained in this work in 0.1 M potassium phosphate, pH 6.0, and 4 °C with enzyme concentrations of 100 µM using a 0.1-cm cuvette. Note that the ordinate scale for B is expanded by a factor of two as compared with the scale for the others. The spectra for the ferrous derivatives for NOS below 320 nm (B-D) and for P-450-CAM below 360 nm (B) are omitted because of optical interference by large amounts of dithionite used. See the legend to Fig. 1and ``Experimental Procedures'' for the details for conditions and sample preparations for NOS.



Comparison of the Active Site Heme Environments of NOS, P-450-CAM, and Chloroperoxidase Based on Their CD Spectral Differences

Although an isolated heme chromophore exhibits no optical activity because of its high symmetry (C or C), it becomes optically active when bound in an asymmetric protein environment(19) . According to the theoretical analysis by Hsu and Woody (42) of the positive single-banded Soret CD spectra of sperm whale Mb and horse Hb, the optical activities arise predominantly from dipole-dipole couplings originating from interactions of the -* transitions of the heme and of nearby aromatic amino acids. However, such a theoretical analysis has not been done for many other heme proteins, and phenomenological spectral diagnoses have often been used for qualitative structural interpretations(19) .

The crystal structure of P-450-CAM revealed that its active site heme environment is composed of mostly nonpolar amino acid residues(43) . Tyr and Thr (a conserved residue for P-450s) are the only polar groups directly surrounding the heme. Significantly, P-450s lack a distal His and another polar group (such as Arg), a pair of which are considered to be important for peroxidase catalysis(43) . Moreover, the known tertiary structures of several peroxidases and of P-450-CAM differ (43) . Chloroperoxidase has a peroxidase-type polar heme environment (26) and an active site topology different from that of P-450-CAM(44) . It is thus not necessarily unexpected that P-450-CAM and chloroperoxidase exhibit totally different Soret CD band patterns as shown in Fig. 5. On the other hand, the Soret CD spectra of NOS and P-450-CAM appear to differ only in sign but are similar in shape.

Certain His-ligated monomeric Hbs such as soybean leg Hb and lamprey Hb exhibit negative Soret CD bands while mammalian Mbs have positive bands (19) . Yet, their tertiary structures are similar, except that leg Hb (as well as lamprey Hb) has a broader heme distal site than the Mbs (45) . Interestingly, binding of alkyl isocyanides to deoxy-ferrous lamprey Hb converts its negative Soret CD band to a positive band in a manner that is dependent on the length (hydrophobicity) of the alkyl group(19) . Thus, the sign of the Soret CD bands of heme proteins appears to be sensitive to the size and/or hydrophobicity of the heme environment. The Soret CD band sign also changes upon binding of SH (but not alkyl- or aryl-thiolates) to the heme iron of ferric P-450-CAM (negative to positive sign) (39) or upon flipping of the heme by 180° about an inplane axis in ferrous-CO sperm whale Mb (positive to negative sign)(46) .

Thus the mirror image Soret CD bands for NOS and P-450-CAM may be attributed to likely differences in size or hydrophobicity of their active sites heme environments. Without having tertiary structural information available for NOS, however, it is not possible at present to assess the size of its active site in relation to that of P-450-CAM. Nevertheless, the heme environment of NOS is likely to be more polar than that of P-450-CAM since NOS binds a highly polar substrate, L-Arg.

Conclusions

The fingerprinting capability of MCD spectroscopy has been used to probe the active site heme coordination structure of native rat brain neuronal NOS. The spectra of several derivatives of ferric and ferrous NOS (the resting, L-Arg-bound ferric, and deoxy-ferrous catalytic intermediates, and the CO- and NO-inhibited ferrous adducts) have been compared with those of corresponding derivatives of P-450-CAM and chloroperoxidase, two known thiolate-ligated heme proteins(20) . The resulting data provide compelling evidence for endogenous thiolate (cysteinate) ligation to the heme iron of NOS. This conclusion is consistent with the results from recent site-directed mutagenesis studies of rat brain neuronal (47, 48) and human endothelial (49) NOSs. Furthermore, the heme-related Soret CD bands for NOS and P-450-CAM, the latter of which represents P-450s in general, have been found to be similar in shape but almost mirror images. This suggests that the active sites of NOS and P-450 may share some common structural features, but significant distinctions exist between their heme environments in certain factors such as hydrophobicity or size.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants CA-53914 (to D. J. S.), GM-51588 (to M. I.-S.), and GM-26730 (to J. H. D.), and by a grant-in-aid from the American Heart Association (to M. I.-S.). The Jasco J-500 spectrometer was purchased with National Institutes of Health Grant RR-03960, and the electromagnet was obtained with a grant from the Research Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: NO, nitric oxide; NOS, nitric oxide synthase; H(4)biopterin, (6R)-5,6,7,8-tetrahydrobiopterin; P-450, cytochrome P-450; P-450-CAM, camphor-hydroxylating cytochrome P-450 isolated from Pseudomonas putida; P-450, fatty acid-hydroxylating cytochrome P-450 isolated from Bacillus megaterium; P-450, beta-naphtoflavone- or 5,6-benzoflavone-inducible cytochrome P-450 isolated from rabbit liver microsomes; Mb, myoglobin; Hb, hemoglobin; MCD, magnetic circular dichroism; Bis-Tris, bis(2-hydroxyethyl)-iminotris(hydroxymethyl)-methane.

(^2)
D. J. Stuehr, unpublished results.

(^3)
A. Matsuoka and M. Ikeda-Saito, unpublished results.

(^4)
Other bacterial P-450s including P-450 and P-450 (P-450 isolated from alpha-terpineol-oxidizing Pseudomonad) also exhibit negative Soret CD bands very similar to those of P-450-CAM(50) .


ACKNOWLEDGEMENTS

We thank Profs. Lowell P. Hager for a kind gift of purified chloroperoxidase and Laura A. Andersson for communicating CD data for P-450 and P-450 prior to publication and Drs. Edmund W. Svastits and John J. Rux for developing the computer-based spectroscopic data-handling system.


REFERENCES

  1. Feldman, P. L., Griffith, O. W., and Stuehr, D. J. (1993) Chem. Eng. News 71 (51),26-38
  2. Ignarro, L. J. (1990) Annu. Rev. Pharmacol. Toxicol. 30,535-560 [CrossRef][Medline] [Order article via Infotrieve]
  3. Nathan, C. (1992) FASEB J. 6,3051-3064 [Abstract/Free Full Text]
  4. Stuehr, D. J., and Griffith, O. W. (1992) Advan. Enzymol. Relat. Areas Mol. Biol. 65,287-346 [Medline] [Order article via Infotrieve]
  5. Marletta, M. A. (1993) J. Biol. Chem. 268,12231-12234 [Free Full Text]
  6. Bredt, D. S., and Snyder, S. H. (1994) Annu. Rev. Biochem. 63,175-195 [CrossRef][Medline] [Order article via Infotrieve]
  7. Stuehr, D. J., Kwon, N. S., Nathan, C. F., Griffith, O. W., Feldman, P. L., and Wiseman, J. (1991) J. Biol. Chem. 266,6259-6263 [Abstract/Free Full Text]
  8. Ravichandran, K. G., Boddupalli, S. S., Hasemann, C. A., Peterson, J. A., and Deisenhofer, J. (1993) Science 261,731-736 [Medline] [Order article via Infotrieve]
  9. White, K. A., and Marletta, M. A. (1992) Biochemistry 31,6627-6631 [Medline] [Order article via Infotrieve]
  10. Stuehr, D. J., and Ikeda-Saito, M. (1992) J. Biol. Chem. 267,20547-20550 [Abstract/Free Full Text]
  11. McMillan, K., Bredt, D. S., Hirsch, D. J., Snyder, S. H., Clark, J. E., and Masters, B. S. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,11141-11145 [Abstract]
  12. Wang, J., Stuehr, D. J., Ikeda-Saito, M., and Rousseau, D. L. (1993) J. Biol. Chem. 268,22255-22258 [Abstract/Free Full Text]
  13. Wang, J., Rousseau, D. L., Abu-Soud, H. M., and Stuehr, D. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,10512-10516 [Abstract/Free Full Text]
  14. Ortiz de Montellano, P. R. (1986) in Cytochrome P-450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., ed) pp. 217-271, Prenum Press, New York
  15. Guengerich, F. P., and MacDonald, T. L. (1990) FASEB J. 4,2453-2459 [Abstract/Free Full Text]
  16. Sono, M., and Dawson, J. H. (1994) in Encyclopedia of Inorganic Chemistry (King, R. B., ed) Vol. 4, pp. 1661-1682, John Wiley & Sons, Chichester, U. K.
  17. Pufahl, R. A., Wishnok, J. S., and Marletta, M. A. (1995) Biochemistry 34,1930-1941 [Medline] [Order article via Infotrieve]
  18. Dawson, J. H., and Dooley, D. M. (1989) in Iron Porphyrins (Lever, A. B. P., and Gray, H. B., eds) pp. 1-135, VCH Publishers, New York
  19. Myer, Y. P., and Pande, A. (1978) in The Porphyrins (Dolphin, D., ed) Vol. III, pp. 271-322, Academic Press, New York
  20. Dawson, J. H., and Sono, M. (1987) Chem. Rev. 87,1255-1276
  21. Sono, M., Hager, L. P., and Dawson, J. H. (1991) Biochim. Biophys. Acta 1078,351-359 [Medline] [Order article via Infotrieve]
  22. Paul, K. G., Theorell, H., and Akeson, A. (1953) Acta Chem. Scand. 7,1284-1287
  23. Huff, A. M., Chang, C. K., Cooper, D. K., Smith, K. M., and Dawson, J. H. (1993) Inorg. Chem. 32,1460-1466
  24. Gunsalus, I. C., and Sligar, S. G. (1978) Advan. Enzymol. Relat. Areas Mol. Biol. 47,1-44 [Medline] [Order article via Infotrieve]
  25. White, R. E., and Coon, M. J. (1982) J. Biol. Chem. 257,3073-3083 [Abstract/Free Full Text]
  26. Sono, M., Dawson, J. H., Hall, K., and Hager, L. P. (1986) Biochemistry 25,347-356 [Medline] [Order article via Infotrieve]
  27. Andersson, L. A. (1982) The Active Site Environments of Heme Mono-oxygenases: Spectroscopic Investigations of Cytochrome P-450 and Secondary Amine Mono-oxygenase , Ph. D. thesis, University of South Carolina
  28. Vickery, L., Salmon, A., and Sauer, K. (1975) Biochim. Biophys. Acta 386,87-98 [Medline] [Order article via Infotrieve]
  29. Dawson, J. H., Trudell, J. R., Barth, G., Linder, R. E., Bunnenberg, E., Djerassi, C., Chiang, R., and Hager, L. P. (1976) J. Am. Chem. Soc. 98,3709-3711 [Medline] [Order article via Infotrieve]
  30. Dawson, J. H., Holm, R. H., Trudell, J. R., Barth, G., Linder, R. E., Bunnenberg, E., Djerassi, C., and Tang, S. C. (1976) J. Am. Chem. Soc. 98,3707-3709 [Medline] [Order article via Infotrieve]
  31. McMillan, K., and Masters, B. S. S. (1993) Biochemistry 32,9875-9880 [Medline] [Order article via Infotrieve]
  32. Matsuoka, A., Stuehr, D. J., Olson, J. S., Clark, P., and Ikeda-Saito, M. (1994) J. Biol. Chem. 269,20335-20339 [Abstract/Free Full Text]
  33. Peterson, J. A. (1971) Arch. Biochem. Biophys. 144,678-693
  34. Tsai, R., Yu, C.-A., Gunsalus, I. C., Peisach, J., Blumberg, W., Orme-Johnson, W. H., and Beinert, H. (1970) Proc. Natl. Acad. Sci. U. S. A. 66,1157-1163 [Abstract]
  35. O'Keeffe, D. H., Ebel, R. E., and Peterson, J. A. (1977) J. Biol. Chem. 253,3509-3516 [Medline] [Order article via Infotrieve]
  36. Sono, M., and Dawson, J. H. (1984) Biochim. Biophys. Acta 789,170-187 [Medline] [Order article via Infotrieve]
  37. Dawson, J. H., Andersson, L. A., and Sono, M. (1983) J. Biol. Chem. 258,13637-13645 [Abstract/Free Full Text]
  38. Sono, M., Eble, K. S., Dawson, J. H., and Hager, L. P. (1985) J. Biol. Chem. 260,15530-15535 [Abstract/Free Full Text]
  39. Andersson, L. A., Sono, M., and Dawson, J. H. (1983) Biochim. Biophys. Acta 748,341-354 [Medline] [Order article via Infotrieve]
  40. Chiang, Y.-L., and Coon, M. J. (1979) Arch. Biochem. Biophys. 195,178-187 [Medline] [Order article via Infotrieve]
  41. Shimizu, T., Iizuka, T., Mitani, F., Ishimura, Y., Nozawa, T., and Hatano, M. (1981) Biochim. Biophys. Acta 669,46-59 [Medline] [Order article via Infotrieve]
  42. Hsu, M.-C., and Woody, R. W. (1971) J. Am. Chem. Soc. 93,3515-3525 [Medline] [Order article via Infotrieve]
  43. Poulos, T. L. (1988) Adv. Inorg. Biochem. 7,1-36
  44. Samokyszyn, V. M., and Ortiz de Montellano, P. R. (1991) Biochemistry 30,11646-11653 [Medline] [Order article via Infotrieve]
  45. Ollis, D. L., Appleby, C. A., Colman, P. M., Cutten, A. E., Guss, J. M., Venkatappa, M. P., and Freeman, H. (1983) Aust. J. Chem. 36,451-468
  46. Aojula, H. S., Wilson, M. T., Moore, G. R., and Williamson, D. J. (1983) Biochem. J. 250,853-858
  47. Richards, M. K., and Marletta, M. A. (1994) Biochemistry 33,14723-14732 [Medline] [Order article via Infotrieve]
  48. McMillan, K., and Masters, B. S. S. (1995) Biochemistry 34,3686-3693 [Medline] [Order article via Infotrieve]
  49. Chen, P.-F., Tsai, A.-L., and Wu, K. K. (1994) J. Biol. Chem. 269,25062-25066 [Abstract/Free Full Text]
  50. Andersson, L. A., and Peterson, J. A. (1995) Biochem. Biophys. Res. Commun. 22,389-395

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