©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Modulation of the Heme Environment of Neutrophil Cytochrome b to a Cytochrome P450-like Structure by Pyridine (*)

(Received for publication, October 17, 1994; and in revised form, November 28, 1994)

Hirotada Fujii (1)(§) Takashi Yonetani (2)(¶) Toshiaki Miki (1) Katsuko Kakinuma (1)

From the  (1)Department of Inflammation Research, The Tokyo Metropolitan Institute of Medical Science, 18-22, Hon-komagome 3-chome, Bunkyo-ku, Tokyo 113, Japan and the (2)Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effect of pyridine on the heme environment of cytochrome b was studied using ESR and optical absorption spectroscopy in relation to the O(2)-generating activity in the NADPH oxidase system of stimulated pig neutrophils. As the concentration of pyridine increased, the absorption maxima of the alpha- and -bands of cytochrome b shifted which correlated with a concomitant decrease in O(2)-generating activity. In addition, the g = 3.2 signal of cytochrome b decreased with the concomitant appearance of a new ESR spectrum that strikingly resembled that of cytochrome P450. The results suggest that pyridine induces a structural modification in the heme environment of cytochrome b by shifting the 5th heme ligand (histidine) to a nearby thiolate group without direct binding of pyridine to the heme. The existence of a reactive thiolate near the heme iron was confirmed by pretreatment of blocked cytochrome b with p-chloromercuribenzoate, which completely inhibited the formation of the cytochrome P450-like ESR spectrum. The results provide further evidence that a low-spin heme iron of cytochrome b with a g-value of 3.2 is essential to the O(2)-forming reaction of the NADPH oxidase system. From sequence alignments of cytochrome P450 with those of large and small subunits of cytochrome b, the heme in cytochrome b appears to be specifically associated with the large subunit.


INTRODUCTION

A phagocyte-specific cytochrome b is an essential component of the membrane-bound NADPH oxidase system which produces superoxide anions (O(2)) in response to invading microorganisms (for reviews, see (1) and (2) ). The functional importance of the phagocytic NADPH oxidase as well as the critical role of cytochrome b were exemplified from studies of chronic granulomatous disease (3, 4, 5) from extensive detailed immunological and biological studies of cytochrome b(6, 7, 8) .

Cytochrome b is a membrane-bound heterodimer of a 91-kDa glycoprotein (6, 7) and a 22-kDa subunit(7, 8) . The amino acid sequences of the large and small subunits of cytochrome b were determined from the corresponding cDNA (7) . Although detailed primary structure data on cytochrome b have been reported, the location and the function of the heme iron have not been characterized as well. Cytochrome b was thought to behave like the terminal component of an electron transport chain in the NADPH oxidase system because of its low redox potential (9) and from spectroscopic studies(10, 11) , but this has not been proven directly. Pyridine has been shown to inhibit both the respiratory burst in intact neutrophils and NADPH-dependent oxygen consumption in lysed cells(12) . The inhibition was accompanied by a spectral change in reduced cytochrome b at 77 K, suggesting that pyridine binds to the heme iron(12) . However, recently it was reported that pyridine was not an inhibitor of oxygen consumption in a model O(2)-forming system consisting of purified cytochrome b plus an exogenous flavoenzyme(13) . After considering these contrary reports, it became clear that it was important to confirm whether pyridine binds directly to the heme iron of cytochrome b, and if it inhibited the NADPH oxidase system in neutrophils.

Recently, we assigned the g = 3.2 ESR signal of the low-spin heme iron of cytochrome b(14, 15) , which was similar to the mitochondrial b-type cytochromes. In this study, we further examine the effects of pyridine on the O(2)-forming activity of the NADPH oxidase from solubilized membranes of stimulated neutrophils in relation to modifications of the heme environment of cytochrome b using ESR and absorption spectroscopy. We observed a dose-dependent inhibitory effect of pyridine on the oxidase activity which was precisely linked to both visible and ESR spectral changes, suggesting that pyridine induced a modification of the heme environment, but without binding directly to the heme iron. Unexpectedly, we found a new ESR signal in pyridine-treated cytochrome b, which was very similar to that of cytochrome P450, providing a clue to the location of the heme.


EXPERIMENTAL PROCEDURES

Materials

DEAE-Sepharose CL-6B and heparin-Sepharose 6B were obtained from Pharmacia Biotech Inc. Heptylthioglucoside (HTG) (^1)was purchased from Dojindo Laboratories, Kumamoto, Japan. NADPH was purchased from Oriental Yeast, Tokyo. Sodium myristate, diisopropyl fluorophosphate and pyridine were from Wako Pure Chemicals Co., Tokyo. Superoxide dismutase and cytochrome c (type VI, from horse heart) were purchased from Sigma. p-Chloromercuribenzoic acid sodium salt (PCMB) was purchased from Nakarai Chemicals. Other chemical reagents were of analytical grade.

Preparation of Membrane Vesicles from Stimulated Neutrophils

Neutrophils were obtained from pig blood(16) . Stimulated cells were prepared with sodium myristate, and the resulting cells were disrupted by sonication(17) . The post-nuclear supernatant was separated from the cell sonicate and used for collection of membrane vesicles.

Solubilization of the NADPH Oxidase

The membrane-bound NADPH oxidase of the stimulated cells was solubilized at 0 °C with 1% HTG, 30% glycerol, 50 mM phosphate buffer, pH 7.0, as reported previously(18) . Treatment of stimulated membrane vesicles with HTG resulted in a high yield (95-100%) of solubilized oxidase.

Purification of Cytochrome b

Cytochrome b was 99% solubilized with HTG from membrane vesicles as described above(18) . The soluble fraction prepared from resting neutrophils was passed through a DEAE-Sepharose CL-6B column (5-ml bed volume) and then applied at a flow rate of 0.5 ml/min to a heparin-Sepharose column (10 ml of resin) equilibrated with a buffer composed of 50 mM phosphate buffer, pH 7.0, 50 mM NaCl, 10% glycerol, and 0.6% HTG(15, 19) .

Assay of O-generating Activity

NADPH-dependent O(2)-generating activity was measured as the rate of cytochrome c reduction in the absence of superoxide dismutase minus the rate with superoxide dismutase in the assay medium of 1.5 mM MgCl(2) and 50 mM phosphate buffer, pH 7.0. The reaction was started by the addition of 0.1 mM NADPH, and the increase in the absorption rate at 550-540 nm was followed at 25 °C in a Hitachi 556 (double beam) spectrophotometer.

Pyridine Inhibition of O-generating Activity of NADPH Oxidase

The reaction mixture (800 µl) contained the solubilized NADPH oxidase (30 µg of protein/ml) prepared from stimulated neutrophils, 30 µM cytochrome c, and 50 mM phosphate buffer, pH 7.0. Pyridine was added into the above mixture and incubated for 0-60 min before addition of 0.1 mM NADPH. The percent inhibition was calculated from the ratio of activity with and without pyridine.

Absorption Spectroscopy

Absorption spectra were recorded at 25 °C in a Unisoku Biospectrometer (model US-401).

ESR Measurements

ESR spectra were measured at 10 K with a Jeol (JES-FE) X-band ESR spectrometer equipped with a Heli-Tran cryostat system (Air Products, model LTR-3). Typical conditions were: microwave power, 10 mW; modulation amplitude, 6.3 G at 100 KHz; response, 0.3 s; sweep time, 4 min; temperature, 5-20 K. For ESR spectrometry of the solubilized oxidase and purified cytochrome b, the sample preparations were concentrated by centrifugation with Amicon Centricon-30 at below 5 °C.

Analysis of Amino Acid Sequences

Amino acid sequence alignments were performed using the program (IDEAS) and protein data bases (PIR) of the Tokyo Metropolitan Institute of Medical Science.


RESULTS

Fig. 1shows the inhibitory effects of pyridine on the O(2)-generating activity of solubilized NADPH oxidase from stimulated neutrophils with time of incubation. An inhibition of O(2)-forming activity was observed with increasing pyridine concentrations, which reached to a plateau after a relatively short incubation time, 10 min. (50% inhibition at 30-50 mM pyridine after 10 min incubation.) Relatively high concentrations of pyridine were required to inhibit the O(2)-generating activity effectively, suggesting that the effect was possibly due to a structural modification of cytochrome b, rather than the simple binding of pyridine to the heme iron.


Figure 1: Time course of the inhibitory effect of pyridine on the O(2)-generating activity of the solubilized NADPH oxidase prepared from stimulated cells. NADPH oxidase was incubated over varying time periods in the presence of increasing concentrations of pyridine, prior to the O(2)-generating activity assay. The concentrations of pyridine used were 12.5 mM (a, circle), 75 mM (b, bullet), 125 mM (c, down triangle), and 150 mM (d, ).



Absorption spectra of the solubilized oxidase were measured in the presence of increasing pyridine at 25 °C. Difference spectra of dithionite-reduced minus oxidized oxidase are shown in Fig. 2. In the absence of pyridine, cytochrome b of the solubilized oxidase shows alpha- and -bands at 559 and 427 nm, where no remarkable changes were observed up to 50 mM pyridine (trace c in Fig. 2). Upon increasing the pyridine concentration up to 500 mM, the peaks shifted to 556 and 422 nm, respectively, accompanied by a decrease in peak intensities (traces d and e in Fig. 2). At 50 mM pyridine (Fig. 2, trace c), a slight shift occurs in the -band. Prolonged incubation (over 10 min) produced no additional changes in the difference spectra, which was similar to that observed for the inhibition by pyridine on O-forming NADPH oxidase activity (Fig. 1).


Figure 2: Effect of pyridine on the difference spectra of cytochrome b in the solubilized NADPH oxidase. Solubilized oxidase (30 µg of protein) was preincubated for 10 min in 50 mM phosphate buffer, pH 7.0, containing varying amounts of pyridine: 0 (trace a), 10 (trace b), 50 (trace c), 100 (trace d), and 500 mM (trace e). Solid dithionite was added prior to measuring the difference spectra.



Since at quite high concentrations of pyridine, the changes in the absorption spectra of cytochrome b were very small, we investigated if ESR spectrometry could yield information on the spin state and its environment of the heme iron. ESR spectra of solubilized oxidase (corresponding to 48 µM cytochrome b) were measured with increasing pyridine concentration at 10 K (Fig. 3). In the absence of pyridine, the g = 3.2 ESR signal, assigned to the low-spin heme of cytochrome b, was prominent (Fig. 3, trace a)(14, 15) . Another low-spin ESR signal was also observed at g = 3.0(14) , which was tentatively assigned to a different state of cytochrome b. The g = 3.2 signal decreased with the concomitant appearance of new ESR signals at g-values of 1.9-2.4, when the concentration of pyridine was increased to 0.5 M (Fig. 3, trace d). (Note that the pH of the sample unchanged by pyridine addition.) The new ESR signals were strikingly similar to those reported for cytochrome P450(20, 21) . In order to confirm that the new ESR signals in Fig. 3, trace d, were attributable to a pyridine-modified form of cytochrome b, purified cytochrome b was measured at various concentrations of pyridine. Fig. 4, traces A and B, show typical ESR for 65 µM cytochrome b in the absence and presence of 0.5 M pyridine, respectively. In the absence of pyridine, only the g = 3.2 signal, but not the g = 3.0 signal, was observed (Fig. 4, trace A). In the presence of pyridine, the g = 3.2 signal disappeared and new ESR signals appeared at g-values of 2.36, 2.25, and 1.91 (Fig. 4, trace B), which were similar to those as seen in Fig. 3, trace d.


Figure 3: Effect of pyridine on the ESR spectra of cytochrome b in solubilized oxidase prepared from stimulated cells. ESR spectra of the concentrated solubilized oxidase containing 48 µM cytochrome b measured at 10 K in the presence of various concentrations of pyridine: 0 (trace a), 100 (trace b), 200 (trace c), and 500 mM (trace d). ESR instrumental conditions were: microwave power, 10 mW; modulation, 6.3 G at 100 KHz; response time, 0.3 s.




Figure 4: Effect of pyridine on the ESR spectra of purified cytochrome b in the absence and presence of PCMB. Purified cytochrome b (65 µM) was first measured without pyridine (trace A). Then the same preparation was incubated for 10 min in the absence (trace B) or presence (trace C) of 2 mM PCMB followed by addition of 0.5 M pyridine. ESR instrumental conditions were identical to those in Fig. 3.



The results shown above suggest that the heme structure in cytochrome b is modulated by pyridine and becomes similar to that of cytochrome P450, suggesting that the 5th ligand of the heme in pyridine-modified cytochrome b has been replaced with a thiolate group present in the vicinity of the heme. In order to examine whether such a reactive thiolate group also exists in cytochrome b, the sulfhydryl reagent, PCMB, was employed as a blocking for reactive thiolate cysteine groups. Purified cytochrome b was first treated with 2 mM PCMB, then mixed with 0.5 M pyridine and measured by ESR at 10 K. The resultant spectrum, depicted in Fig. 4, trace C, shows that ``cytochrome P450-like'' signals induced by pyridine were missing after PCMB treatment and a high-spin ESR signal appeared at g = 6.


DISCUSSION

We have shown that pyridine causes shift in the alpha-band from 559 to 556 nm (Fig. 2) in agreement with the results reported by Iizuka et al.(12) using low temperature spectroscopy. In their report, inhibition of the O(2)-generating activity by pyridine was explained by the displacement of the internal heme ligands by a pyridine molecule resulting in changes in the oxidation-reduction potential of cytochrome b(12) . However, relatively high concentrations of pyridine were required to inhibit the O(2)-generating activity, and no release of hemin was observed. This possibly suggests that the pyridine inhibitory effect is due to a structural (conformational) modification of cytochrome b, instead of a displacement of the internal heme ligands with pyridine.

Evidence for a Thiolate-Heme Ligand

We showed previously that neutrophil cytochrome b is a low-spin hemoprotein with a highly anisotropic ESR signal at g = 3.2 that is strongly suggesting of bishistidine coordination of the heme iron(14, 15) . At pyridine concentrations up to 0.5 M, the g = 3.2 ESR signal intensity decreased with a simultaneous increase in new ESR signals at g-values of 2.36, 2.25, and 1.91 ( Fig. 3and Fig. 4). These ESR signals were very similar to those of cytochrome P450 (g-values of 2.42, 2.23, and 1.91) reported by Omura and Sato (20) and Hashimoto et al.(21) . The ESR spectrum of cytochrome P450 was interpreted in the past to arise from contributions from the cysteine thiolate ligand at the heme iron(20, 21) . We suggest that, in the case of cytochrome b, pyridine binding induces a structural modification of the heme environment involving the replacement of the ``5th'' ligand of the heme with a nearby thiolate group. In model studies, Sakurai et al.(22) showed that a complex of glutathione, hemin, and pyridine exhibited a similar ESR spectrum to that of cytochrome P450 with g-values of 2.38, 2.26, and 1.93. Notably, the ESR spectrum of purified cytochrome b in the presence of 0.5 M pyridine was very similar to that of the glutathione-hemin-pyridine complex, also indicating the possibility of the replacement of the 6th ligand by pyridine. From careful analysis of the ESR spectrum in Fig. 4, trace B, an additional ESR signal was also visible as a shoulder at g = 2.415, which was close to the g-value of 2.42 from the spectrum of cytochrome P450 with thiolate-H(2)O coordination (20, 21) . Thus in the presence of pyridine cytochrome b shows two types of low-spin complexes, one of which has thiolate-pyridine coordination, the other of which has thiolate-H(2)O coordination; both forms were unable to support O(2)-generation in the NADPH oxidase. Also, judging from the fact that the absorption spectrum of pyridine-modified cytochrome b was not similar to that of cytochrome P450, a thiolate-pyridine coordination seems reasonable as the predominant species (Fig. 2).

Location of the Heme

Cytochrome b is an important component of the NADPH oxidase system(1, 2, 3, 4, 5, 6, 7, 8) . Nevertheless, the location of the heme in the subunits of cytochrome b has not yet been identified. Previously, Yamaguchi et al.(23) and Nugent et al.(24) reported that the prosthetic group of cytochrome b was associated with the small subunit. Yet, contrary to these reports, our ESR studies suggest a different localization of the heme in cytochrome b as discussed below. Quinn et al.(25) reported recently that cytochrome b is a multiple heme protein as evidenced by the presence of heme in both the large and small subunits. Pseudomonas putida cytochrome P450 was reported to have conserved residues near Cys with an axial thiolate ligand providing hydrophobic contacts with the heme(26) . Four residues, Phe-X-X-Gly-X-X-X-Cys-Leu-Gly-, near the Cys ligand were found to be conserved in cytochrome P450 from several species(26) . In addition, x-ray crystallographic studies of P. putida cytochrome P450 showed that Phe and Leu bracket Cys and, together with Gln, form a hydrophobic pocket for the thiolate group(26) . Our ESR studies suggest that cytochrome b may have similar residues in the vicinity of a Cys near the axial ligand position of the heme. We compared amino acid sequence alignments of both the large and small subunits of cytochrome b with that of cytochrome P450 (Fig. SI). The comparison (Fig. SI) shows that the large subunit has a region (residues 78-88), which is quite similar to that in the vicinity of the Cys in cytochrome P450. In the cytochrome b large subunit, the corresponding hydrophobic residues, Phe, Gly, Ala, and Tyr, may bracket Cys, which could reside at the 5th coordination position of the heme after conformational changes induced by pyridine, i.e. the replacement of the former 5th ligand of the heme, His, by Cys. There are 6 His residues (101, 111, 115, 119, 209, and 210) near Cys(7) , one of which seems to be replaced with Cys. The ESR spectra of cytochrome b(14, 15) suggest that both the 5th and 6th ligands of the heme are most likely His residues. In the case of cytochrome b, we would predict that the 5th ligand is His, and the 6th ligand is His based on data from point mutation studies (His Arg and His Tyr) of the large subunit of cytochrome b in X-linked chronic granulomatous disease reported by Bolscher et al.(27) . Dinauer et al.(28) reported that there is only one invariant histidine residue in the small subunit (His) from analysis of gene structure in chronic granulomatous disease, we suggest that the small subunit of cytochrome b may not be capable of heme coordination, if bishistidine ligation is required. This hypothesis is also consistent with the fact that analogous residues to those of cytochrome P450 were not found in the small subunit of cytochrome b.


Scheme I: Scheme 1



Last, our previous studies using ESR spin labels showed that the O(2)-releasing site was not located at the outer surface of the neutrophil plasma membrane, but instead in an interior hydrophobic environment, a short distance from the outer surface(29) . Two potential heme iron ligands, namely His and His , are located at an interior hydrophobic region close to the glycosylation sites (Asn, Asn, and Asn) of the large subunit(30) .


FOOTNOTES

*
This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan. A preliminary account of this work was presented at the International Conference on Bio-Radicals Detected by ESR Spectroscopy, Yamagata, Japan (June 12-16, 1994). 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.

§
To whom correspondence should be addressed: Dept. of Inflammation Research, the Tokyo Metropolitan Institute of Medical Science, 18-22, Hon-komagome 3-chome, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3823-2101; Fax: 81-3-3823-2965; fujii{at}rinshoken.or.jp.

Visiting professor from the University of Pennsylvania, Philadelphia, PA 19104.

(^1)
The abbreviations used are: HTG, n-heptyl-beta-thioglucoside; PCMB, p-chloromercuribenzoic acid sodium salt.


ACKNOWLEDGEMENTS

We are very grateful to Prof. Lawrence J. Berliner, The Ohio State University, for critical reading of our manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.