Biochimica et BiophysicaActa, !136(1992)239-246
239
© 1992Elsev;erSciencePublishersB.V.All rightsreserved0167-4889/92/$05.00
BBAMCR13226
Electron paramagnetic resonance studies on cytochrorne b-558 and peroxidases of pig blood granulocytes Hirotada Fujii and Katsuko Kakinuma Department o[ Inflammation Research. The Tokyo ~letropolitan bzstituteof Medical Science, Bunkyo-ku. Tokyo (lapan)
(Received 3 April 1992)
Keywords: Granulocyte;Neutrophil;Cytochromeb-558;EPR;NADPHoxid~Lse;(Pig) Low-temperature electron paramagnetic resonance (EPR) spectrometry on granulocytesprepared from pig blood was carried out with concentrated cellular and subccllelar fractions to characterize EPR signa!s of cytochrome b-558 (.cyt b-558). A thick cell suspension (-- 2.10 '~ cells/ml), c~ntaining mostly neutrophils, showed typical high-spin EPR signals due to myeloperoxidase (MPO) and a low spin signal at a g value of around 3.2. A similar thick granulocyte suspension containing eosinophils showed not ,nly these signals but also low spin heme signals at g values of 2.86, 2,13, and 1.66, which have been reported to be of cyt b-558 (Ueno et al. 1991, FEBS Lett. 281, 130-132). MPO and eosinophil peroxidase (EPO) were rcle~. ,~.dfrom the membrane fractions with 50 mM phospha.,ebuffer {pH 7.0) containing I M NaCt, and then were highlyeoncentratetl, in which no cyt b-558 was detected by absorption spectra. The signal at a g value of 2.86 was found only in the EPO fraction, suggesting that this signal is derived from a low-spin form of an EPO-complex, but neither from MPO nor cyt b-558. The O;-forming NADPH oxidase associated in the membranes was solubilized with heptylthioglucosideat 0°C and concentrated up to 45 tLM q,t b-558 with no modification of the heme moiety confirmed by its Of-generating activityand lack of carbon monoxide-bindingcapacity. Cyt b-558 showed an anisotropic signal at a g value of 3.2 _+0.05, which was cyanide-insensitiveand reducible with reductants. The signal intensity was concentration dependent, suggestingthat the g = 3.2 signal is characteristic of the low-spin heme iron in cyt b-558.
Introduction
Granulocytes, such as neutrophils and eosinophils, respond by generating the superoxide anion (Of) in response to invading microorganisms [1]. This response is essentially due to the activation of a membrane-associated NADPH oxidase system [2], which has been hypothesized to be a multi-component electron transport system consisting of a membrane-bound flavoprotein [3-5], b-type offochrome [6,7], and cytosolic components [8,9]. Since the discovery of the absence of b-type cytochromes in the neutrophils of patients with X-linked chronic granulomatous disease (CGD) [6] that cannot produce 0 2 when stimulated, this cytochrome has been extensively studied as a key enzyme in the Oj-generating system of neutrophi!s. This cytoehrome, called cytochrome bs58 (off b-558), was alc,o found in n~noq, tes, eosinophils, and maerophages [11. Cyt b-558 is a heterodimer consisting of a heavily glycosylated ql-kDa subunit (,8-subunit) and a non-glycosylated 22
Correspondenceto: K. Kakinama,Departmentof InflammationResearch. The TokyoMem'~olitanInstituteof MedicalScience,18.22 Honkomagome3-chorea,P,unlqo-ku,Tokyoi13, Japan.
kDa subunit (a-subunit) [10,11]. The genes for both these molecules have been cloned and sequenced [12,13]. Although detailed structural and immunological data on cyt b-558 have been reported [I,q--13], it is not as well characterized functionall~;. Several researchers have suggested that cyt b-558 is the terminal oxidase of the respiratory electron transport chain due to its unusually low redox potential (E,, ~- -240 mV) [14] and carbon monoxide (CO)-binding capacity [14,15]. However, this viewpoint has been challenged by other observations that cellular cyt b-558 does not bind CO [16]. These contradictory results might be due to structural modification of 'he heine; i.e., solubilization or purification of cyt b-558 has been reported to gi,te rise to increased CO-binding affinity and a large shift in its reduction potential [14,15,17,18], Electron paramagnetic resonance (EPR) spectroscopy on off b-558 has been reported by several groups [19-22]. Previously, an immunoprecipitated fraction from solubilized NADPH oxidase showed cyanide-insensitive high-spin signals, but only in the stimulated preparations [19], which had been considered to be due to off b-558 but were later found to be due to modified myeloperoxidase (MPO) [20]. Recent
240 EPR studies of a solubilized fraction from neutrophil membranes containing 11 ,aM cyt b-558 showed no heine signals, except that from high-spin MPO [21]. On the other hand, low-spin heine signals at g values of 2.85, 2.21 and 1.67 were recently found for 03 b-558 in a thick neutrophil suspension [22]. in order m characterize the EPR spectma of cyt b-558, it is critical to prepare a highly concentrated 03 b-558 sample, with little contamination from other hemoproteins, and with no modification of the 03 b-558 heme moiety. In the present work, we first examined the heme proteins, 03 b-558, MPO and eosinophil peroxidas¢ (EPO), at both cellular and subeellular levels, to distinguish their EPR signals. A low spin heine signal detected in concentrated cyt b-558 fractions, probably derived from the heme iron of cyt b-558, is discussed, based on spectrophotometric results obtained for all the cellular and subcellular samples. Materials and methods
Materials The fatty acids, myristic acid and arachidonic acid, were from Wako Pure Chemical, Tokyo, and were dissolved in ethanol as descrl3md previously [23]. Heptylthioglucoside (HTG) was purchased from Dojindo Laboratories, Kumamoto. NADPH and NADH were from Oriental Yeast, Tokyo. 5uperoxide dismutase (SOD), cytochrome c (type VI, from horse heart), and glucose oxidase were purchased from Sigma. Other reagents were of analytical grade.
Preparation of gramdocytes Granulocytes were obtained from pig blood as reported previously [5,24] by the Conray-Ficoli differential density centrifugation method. The erythrocytes contaminated in the huffy coat were hemolyzed with a large volume of ice-cold 0.2% NaCI solution for 30 s, and then the preparations were promptly mixed with an equal volume of ice-cold 1.6% NaCI solution to restore the isotonicity. The resulting cell preparation contained about 90% neutrophils and other cells, in which the percent of eosinophils varied. Cell populations were determined by microscopy. Stimulated and unstimulated cells were prepared with and without myristate, as descn'bed previously [25] and were then collected by centrifugation, mixed with ice-cold 0.34 M sucrose containing 50 mM phosphate buffer (pH 7.4) at 2" l0 s cells/ml and promptly frozen, as described previously [25].
of neutrophils and eosinophils, respectively. For neutrophils, the soaication time was set at 6 x !.5 s at 20 W with intervals of ! s. The sonicated suspension, which contained more intact eosinophils than new trophils, was centrifuged at 500 x g for 5 rain at 0-2°C to remove unbroken cells and nuclei. The post-nuclear supernatant was centrifuged at 100000 x g for 60 rain at 0 - T C to collect membrane vesicles containing the NADPH oxi-dase and azurophil granules, as membrane fraction l. For eosinophils, the sonication time was set at 30 s ( 1 5 x 2 s) with intervals of 1 s and then centrifuged in the san~.e manner to obtain membrane vesicles containing the oxidase, azurophil and eosinophii granules, as membrane fraction II. Azurophil and eosinophil granules were detected spectrophotometrically as described below.
Separation of MPO and EPO from membranefractions Membrane fraction ! (I-10 ~° cell equivalent) obtained by the above procedure was mixed with 100 ml ,,,f 50 mM phosphate buffer containing 1 M NaCI at 0-2°C for ! h, and then centrifuged at 100000 × g for 30 rain at 0-2~C. The resulting supernatant contained fairly large amounts of MPO. The EPO-rich fraction was separated from the Membrane fraction il by the treatment with the same NaG-containing buffer and centrifugation in the same manner. The two supernatants were concentrated with Amicon centricone-30 microconcentrator by centrifugation. Concentrations of MPO and EPO were determined spectrophotometrically using the absorbance coefficients of the Soret bands: 89 cm -I mM at 428 nm for MPO [26] and 110 cm -t mM at 412 nm for EPO [27].
Solubilization of O't b-558 The membrane-associated 03 b-558 was solubilized at if'C, as described previously [28]. The membrane fraction 1 obtained by the above procedure was suspended a~: a final concentration of :~ mg protein per ml in a solubilizing mixture consisting of I% (w/v) heptylthioglucoside (HTG), 30% glycerol and 50 mM phosphate buffer (pH 7.0). The mixture was gently stirred in an ice-bath for 30 rain and then centrifuged at 100000 x g for 60 rain to obtain the solubilized ojt b-558 supematant. The cytochrome rich fraction was concentrated with AmJcon centricone-30 microconcentrators by centrifugation at 5000 x g for 10 h at 0 - 5 ~ , which concentration was dete .rmined from the difference s ~ r a of dithionite reduced minas oxidized samples, using the molar extinction coefficient of 21.6-103 (558 minus 540 rim) [29].
Destruction of neutrophils and eosinophils The frozen cells (1-10 l° cells/50 ml) were thawed and sonicated at {PC by two different methods in a Branson Sonifier (model 185) equipped with a controller to set different sonieation times for destruction
Spectrophotomelric measurements Absorption spectra were measured at 25°C in the range of 400 and 600 or 700 nm in a microcuvette (10 mm light path, 3 mm width) in a Unisoku Biospec-
241 trophotometer US-401 (Unisoku, Osaka), controlled by a computer, NEC PC9801.
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CO-bbuling experiment The reduced minus oxidized difference spectra of cyt b-558, either with NADPH or dithionitc, were measured with and without CO over 400 and 600 nm in a microcuvette (10 turn light path and 3 mm width) containing a reaction mixture consisting of 60 #i of the solubilized sample and an equal volume of 0.I M phosphate buffer (pH 7.0). The concentration of cyt b-558 used was 2 to 3 pM. Strict anaerobic conditions were aeh,~eved according to the reported method [28] using pure argon and a system of glucose and glucose oxidase. For CO difference spectra, a NADPH or dithionite-reduced solubilized sample was equilibrated with o~gen-free CO of purity greater than 99.95% for 15 rain at 4°C. ;
NADPH dependent O2-generating activity was measured p~ the rate of cytochrome c reduction in the absence of SOD, minus the rate with SOD, in a Hitachi 556 spectrophotometer [2].
1 1000
EPR spectra EPR spectra were recorded in a Jeol (JES-FE) X-band EPR spectrometer equipped with LTR HeliTran liquid helium transfer refrigerator (Air Products and Chemicals). The conditions for measurements were as follows: microwave power, 5 roW; modulation amplitude, 6.3 G at 100 KHz; response, 0.3 s; sweep time, 4 min, temperature, 5 to 15 K. For EPR spectrometry of cells, a thick cell sample ((1.8-2.3). 109 cells/hal) was obtained by centrifuging a cell suspension (5. l0 s cells/ml) at 200 × g for 5 rain at 15°C, and was put into an EPR tube (4 mm i.d.). For measurements of EPR spectra of the peroxidases, the MPO or EPO fraction concentrated at 50 to 70 p,M as descn'bed above was transferred into the EPR tu~e, respectively. For the measurement of EPR spectrcmetry of the cyt b-558 fraction, the solubilized cyt b-558 fraction was concentrated and transfe~Ted into the EPR tube at a concentration of 10 m 45 # M cyt b-558. Itemlts and Discassien
EPR spectra of oanulocytes The EPR spectrum of granulocytes (1.8-109 cells/mi) at 10 K is depicted in Fig. 1A, which shows a homogeneous high-spin heine iron spectrum with rhombic symmetry. The g values of 7.03, 5.20 and 1.94 are identical to those of MPO [30]. In addition, low-spin heme signals with g values at 2.86, 2.13 and 1.66 were detected as well as a new unassigned broad signal around g -- 3.2. The EPR spectrum of another sample
151
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Field (gauss) Fig~ !. EPR spectra of pig blood granulocytes.(A) Granulocyles (1.8-109 cells/ml);B: Granulocytes(23.109 ceils/ml);(C) grauulocytcs in (A) treatedwith 2 mMpotassiumcyanide.The EPR instrumentalconditionswereas follows:microwavepower.5 mW;modula. tion,6.3 G at 100kHz;responsetime,0.3 s; temperature.10IL
(2.3.109 cells/ml) in Fig. 1B shows the high-spin EPR signal of MPO but not the low-spin signal at g = 2.86 which appeared in Fig. 1A. The concentrations of cyt b-558 in these two samples were about 16.2 and 20.7 /zM in Fig. 1A and B, respectively. Furthermore, this low-spin signal intensity was not dependent vn the cyt b-558 concentration, suggesting that the low-spin heine signal at g = 2.86 is not due to cyt b-558. When the cells in ~:ig. iA were exposed to 2 mM cyanide, the high-spin MPO signals disappeared and new signals due to the MPO-cyanide complex appeared at g values of 2.84, 2.17 and 1.61 (Fig. IC). The ferric low spin signal at g = 2,86 (Fig. IA) was not affected by the addition of cyanide. The signal at gl = 2.86 was dis. tinct from that of the MPO-cyanlde complex (gt = 2.84), but two other signals, g2 and g3, overlapped those of the MPO-cyanide complex. After considering the cyanide insensitivity and the dithionite reducibility, these signals were previously assigned to cyt b-558 by Ueno et al. [22] with similar cell preparations. However, these granulocyte preparations contained not only neutrophils but also eosinophils, which have different types of granules containing various metaloproteins.
242 Thus, we examined the individual lysos~mal iron pro~eins ( ~ 0 aa~ EPO, ;~n membrane fract~:.-._~ ~ f o r e fmal~ assigning the metalopmtein with g~ -- 2.86.
Absorption spectra and EPR spectra of peroxidase-rich
fracture M P O and E P O releasedwith I M NaCI from the membranes were analyzed by absorption and EPR spectrometries.The differenceabsorptionspectrum of a reduced m/nus oxidized MPO fraction is shown in Fig. 2A. The large absorption peaks at 473 and 640 nm are attn'buted ~ to MPO [26], and while the small peaks at 560 nm and :/95 nm can be aun'butezl mainly to MPO, but also to EPO [31], indicating that the _fr_,~ction ~ Fig. 2A contains predominantly MPO and only a small fraction of EPO. On the other hand, this fraction does not contain cyt b-558, since no absorption peak appeared at 425 nm (Fig. 2A) [6]. The highly concentrated (72 p M ) MPO fraction was subjected to EPR spectroscopy as shown in Fig. 2B. The EPR
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FMd (ga,~) Fi~ 3. Sw.ctmsce~ pmpe~s of the EPO f."-~fion.CA)Difference spectrum of dithim~-reduced n~us oxk!mM state of the EI'O ~ The com~enttationsof EPO and MPO gere 66 ~M and ]6 ~M, n:~ectively. (B) EPR sigch'ma of the EPO haction. (C) EPR speannn of the EI'O ~ treated with 2 mM cyanide recorded at l0 K. The insmnnentai conditions for (13)and (C) ~ere identical to those in ~g. 1.
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Field (gauss) F'~ 2. S p e c t ~ properties of the MPO fraction. (A) Difference ebsorptionspectremof dithionite-reducedminusoxidizedMI'O fraction. The concentration of MPO in the fractionwas 72 pM. (B) EPR spectrum of the oxidized state of the MPO fraction. (C) EPR stpectrum of the MPO fraction treated with 2 mM c/ankle. Instrumental conditio,s for (B) ancl(C}were identical to those in Fig. 1.
spectrum shows high-spin ferric heine signals with rhombic symmetry (g values of 7.02, 5.22 and 1.96), consistent with the previously reported spectrum of MPO [30]. No other EPR signals were detected (Fig. 2B). Addition of 2 mM cyanide to the MPO fraction resulted in the disappearance of the high spin signals with a concomitant appearance of new MPO-cyanide complex signals at g = 2.84, 2224 and 1.66 (Fig. 2 0 . We also examined an EPO-rich fraction (see Materials and Methods). The difference absorption spectrum is shown in Fig. 3A, with typical EPO peaks at 450 rim, 563 nm and 598 nm [31]. The additional band at 640 .,un is due to either EPO or MPO; the large
243 band at 470 nm is due to MPO. Using reported extinction coefficients of EPO [27] and MPO [26], the concentrations of EPO and MPO in this fraction were calculated to be 66 pM and 16 pM, respectively. Again, no cyt b-558 was detected in this highlyconcentrated fraction due to the lack of a peak at 425 nm (Fig. 3A) [6]. The EPR spectrum of the EPO-rich fraction showed both high-spin and low-spin ferric heine signals with g values of 6.86, 5.23 and 1.96, and 2.87, 2.21 and 1.67, respectively.These high-spin heme signals were close to those reported previously with cellular and purified EPO, g = 6.60 [31] and 6.50 [27], respectively.The low-spin heine signals were similar to those for the cells shown in Fig. 1A, Figure 3C shows EPR spectrum of the EPO-rich fTaction treated with 2 mM cyanide. The ferric high-spin heine signals at g = 6.86 and 5.23 (~g. 3B) decreased markedly; instead, the low-spin berne signals from the EPO- and MPO. cyanide complexes appeared at g values of 2.~;3, 2.21 and 1.65. On the other hand, the low-spin signal at g = 2.87 (Fig. 3B) was cyanide-insensitive.Redu.aion of the EPO-rich fraction with sodium dithionite resulted in the disappeazance of both high-spin (g = 6.86 and 5.23) and knv-spin (g = 2.87) heme signals (data not shown). The EPR signal at g = 186-187 (Fig. IA and Fig. 313) has been tentatively assigned to cyt b.558 by Ueno et al. [22]. However, we found that this low-spin ~gnal at g = 186 was sim~ar to that in the cyt b-558free and EPO-rich ~ (Fig. 3B). Therefore this g = 2.86 signal is not from cyt b-558, but is likely due to a low-spin EPO-complex,which might be formed from high-spin EPO with an endogenous fig,and(s)such as OH- and (21- [30]. Furthermore, Ueno et al. [22] obtained a tetragnnal ct~ml field spfitting of 4.96, and thought it to be too large for that of a b-type cytochrome, 3.11-4.25 [32]. However, the value of 4.96 is well within the range calculated for low-spincomplexes of EPO, MPO and catalase (3.66-5.25) [27].
Absorption and EPR spectra of solubilized cyt b-558 Treatment of membrane vesicles with 1% HTG resulted in a high yield of so!ubllizatiou of both cyt b-558 (98.6 + 4.9%, n -- 6) and NADPH oxidase (94.3 + 5.4%, n = 6) in contrast to very slight solub'dization of MPO [28]. Fig. 4 shows difference absorption spectra of dithionite-reduced minus oxidized membrane vesicles (Fig. 4A) and the solubilizcd oxidasc fraction (Fig. 4B), respectively. The peak at 470 nm due to MPO detected in the difference spectrum of membrane vesicles (A) was totally absent in the soinbllized fraction (B), confirming that the solubllized fraction contains negligibleamounts of MPO. On the contrary, the HTG-solnbilizedfraction contained a highly active NADPH oxidase, which produced 250-475 nmol 021nfin per mg of protein (n =6), from my~'istatestimulated cell membraneg while showing negligible
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W~velengLh (rim) Fig. 4. Dithionite-reduced minus oxidized difference spectra of membrane vesicles (A) a,:d the solebilizcd ~.~ b-558 fraction (B). Membra,e vesicles (5 mg protein/m|) and solubilized cyl b-558 fraction (3 mg protein/ml) were prepared in 0.1 M p,hosphate buffer (pH 7.0),
activity with unstimulated cell memb~nes, yet both HTG-solubilized fractions contained almost equal amounts of cyt b-558, 317-333 pmol/mg of protein (n ; 6) [28]. While cyt b-558 hardly binds CO in sit~ [16], the solubilization and isolation from its hydrophobic environment may have resulted in a change in the environment of the heine of cyt 6-558, partly tee,yard .~omething like high-spin ferric heine, consequently increasing its CO-binding capacity, as reported previou~.qy from this laboratory [18].Therefore, wc carefullyexamined the CO-binding capaci~ of HTG-solubilized cyt 6-558. The heme of cyt 6-558 in ~he solubilizedfraction was reduced with 0.1 mM NADPH under anaerobic conditions by incubation for 20 rain [28], and then the reduced minus oxidized difference spectrum wa~ obtained (Fig. 5A). After equilibrating the reduced sample with CO for 15 rain, the difference spectrum was measured (Fig. 5A), Both spectra showed no difference in the a and p bands before and after mixingwith CO. Fig. 5B shows the Co-dlfference spectrum of reduced cyt b-558 fraction in the absence and presence of CO, giving evidence that reduced cyt b-558 in the presence of the physiologicalsubstrate, NADPH, does not bind CO; that is, cyt b-558 may not form an oxygenated complex, consistent with the evidence by lizuka et al. [16]. For comparison, the difference spectra of dithionRe-reduced minus oxidized cyt b-558 were measured
244
15 rain aiIer the incubation with and without CO. The spectra obtained showed that a small portion of reduced cyt b-558 (about 10%) complexed with CO in the dithionite-redueed state (Fig. 5C). In fact, it may he poss~le that dithioni~e might induce this structural modification of the cyt b-558 heine. Overall these r ~ B indicate that the CO-binding capacity of HTGsolubilized cyt b-558 is poor, its binding rate is very slow [33], and the cnordination environment of the solubilized cyt b-558 heine iron with HTG is not modified, as compared with that in cells. The HTG-solobilized sample from either stimulated or resting cell membranes was concentrated to 45 #M cyt b-558. The EPR spectrum is shown in Fig. 6A.
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Field (Eauss) l~y. 6. EPR %oec~raof con~nlrated cyt/7-558fractions from unstimulat~! celk measured at 10 K, (A) EPR spectrum of the highly concentrated fractioncontaining45 p M cyt b-558.(B) 2 raM cyanide was axkled to the cB b-558 baction of (A). (C) Reduction of the cyt =CO
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Wavelength (nm) F*g. 5. Difference absorp~oo spec~ of NADPH-rcduced minus mid~ed state of t~e c!a b-558 fraction in the absence and presence of CO (A, +_CO)and CO-difference spectra of the cyt/7-558fraction reduced with NADPH (9) and dithionite (C). (A) The ojt b-558 fiactioD u)lubilized from stimulated cells in 0,1 M phosphate buffer (pH 7.0) was mixed with an I mM NADPH (final concentration 0.1 raM)anaerobicalb'for 20 rain.The ~ncentmfionof the cyl b-558in
the fractionwas 2 t~M..Afterthe samplewith NADPHwas equilibrated with o'~gen-freeCO (> 99.95%) for 15 min at 4~C, the differencespectrmnwas measured. (B) The CO-differenceb ' ~ m was calculatedfrom the red~'ed spectrawith N A D P H in the presence and absence of CO. (C) The CO~ifferencespectrum was
calculatedfromthe reduced~ecaa withdithionitcin the presence and absenceof CO. The ~ of reducedc~/7-558withdithionite wereraeasured15rainafterthe incubationwithand withoutCO.
/>-558fraction in (A) with dithionite. The instrmnental conditions for (A), (B) and (C) were identicalto those in Fig. I.
Several signals were detected at g values of 7.03, 6.03, 5~3 and 4.23 in the low field region. The g values, 7.~: and 523, were identical to those reported for MPO in the ferric high-spin state [30]. The signal at 4.23 resembles that for non-heine high-spin ferric species. In the high-field region, a distinct EPR signal at a g value of 3.2 + 0.05 was observed. In addition, a small signal was also detected at a g value of about 3.0. Other minor signals could correspond to coppercontaining compounds (g -- 2.08) and o;ganic free radicab (g = ZOO).The EPR spectrum of a similar sample, prepared from stiwulated membranes, was essentially the same as that in Fig. 6A (data not shown). Note that in Fig. 6A, there was no signal at g-- 2.86-2.87, again giving evidence that a g = 2.86-2.87 signal is not due to cyt b-558. Figure 6B shows the EPR spectrum of the cyt b-558 fraction treated with 2 mM cyanide. The ferric high-spin heine signals (Fig. 6A) at g -- 7.03 and 5.23 disappear¢~ while the low-spin heine signals for MPO-cyanide complex appeared at g values of 2.84 and 223, respectively. An unassigned high-spin signal (g -- 6.03) from Fig, 6A also decreased markedly upon cyanide addition. Thus, the high-spin signals at g--
245 7.03, 6.03 and 5.23 were not due to cyt b-558. The low-spin ferric signals at g values of 3.2 and 3.0 did not disappear in either 2 mM cyanide (Fig. 6B) or 4 mM azide (data not shown), yet completely disappeared in dithionite, as shown in Fig. 6C. When the concentrated cyt b-558 fraction was reduced with NADPH under anaerobic conditions, the EPR signals at g = 3.2 and 3.0 diminished (data not shown). The EPR signal ~.ntensityat a g value of 3.2 increased with increasing ey'~ b-558 concentration, but the signal intensity at around g = 3.0 varied in different samples. T h ~ results suggest that the g = 3.2 signal is due mainly to cyt b-558. The other EPR signal at g = 3.0 has not been assigned yet but ~i.~ht ~ due to different state of cyt b-558. The axial amino acid liga::ds to the berne of cyt b-558 have not been identified, but Hurs~ et al. [21] recently reported from resonance raman spectra that the heine is a low-spin six-coordinate with imidazole or imidazolate axial ligation. The g:values of b-type cytochromes are reported to change with the axial ligand; i.e., thiolate-imidazole iigation (g = 2.34-2.46), aliphatic amine-imidazole (g=3.33-3Al), and bis-aliphatic amines (g = 3.57-3.63) [32]. The g = 3.2 observed in this study seems higher than those reported for most b-type cytochromes with bis-histidine coordination, 2.69-3.03 [32]; however, recently, several membranebound b-t3~e cylochromes have been reported to give g values higher than 3.1 [34, 35]. Friden et al. [35] found that Bac/lhts subt///s cytochrome b-558 was a low-spin hemoprotein with a highly anisotropic EPR signal, g = 3.5, and that this cytochrome b-558 was a bis-histidine coordinated hemoprotein, using low teml~erature EPR and magnetic circular dichroism. Thus, the present results, with a highly anisotropic signal at g = 3.~ may be compat~e with b~histidine coordination of neutrophil cyt b-558, as predicted by its resonance raman spectroscopic results [21]. The othcr known g values (e.g., g2 and g3 ) for cyt b-558 were not observed in Fig. 6A, due possibly to overlapping by other signals. Palmer [36] has proposed that if the two imidazole-ring planes of the ligating histidines are gradually rotated from a parallel orientation (g~ = 3.0) toward a perpendicular orientation, the g~ value will increase, while the g2 and g3 components will become progressively smaller. Such a perpendicular orientatio,) of the ligating histidine groups might exist in neutrophil ~ t b-558, resulting in smaller g2 arid g3 components of its EPR spectra. In conclusion, all of these results suggest that the signal at a g value of 33.± 0.05 is derived from cyt b-55g, characteristic for a hexa-coordinated low-spin heme protein, in accord with its CO-insensitivity. Recently, this assignment has been further confirmed from EPR spectra of purified cyt b-558 [37].
Acknowledgements We thank Prof. Lawrence J. Berliner, Department of Chemistry, The Ohio State University, for his helpful discussions and critical reading our manuscript. This work was supported in part by grants from the Ministry of Education, Science and Culture, Japan, and the Naito Foundation. References I Segal, A.W. (1989) J. Clin. Invest. 83. 1785-1793. 2 Babior, B.M., Curnutte, J.T. and McMurrich. BJ. (1976) J. Clio. Invest.58, 989-996. 3 Cross, A.R., Parkinson. J.F. and Jones, O.T.G. (1984) Biochem. J. 223. 337-344. 4 Markert, M.. Glass, G.A. and Babior. B.M. (I985) Prec. Natl. Acad. Sci. USA 82. 3i 14-3148. 5 Kakinuma` K.. Kaneda, M., Chiba` T, and Ohnishi, T. (1986) J. Biol. Chem. 261, 9426-9433. 6 Segal, :LW. and Jones, O.'~".G.{I978) Nature 276. 515-517. 7 Cros,,~,AR.. Parkin~an, J.F. an~ Jones, O.T.G, (1~5) Biechem. J.
226, 881-1~4. 8 BL~omberg.Y. and Pick.E. (1'~4)Cell. lmmunot.88, 213-221. 9 Clark, R.A.. Malech.H.L..Gallin.J.L Nuroi. H.. VOile,B.D, Pearson,D.W.,Nauseef,W.M.and Cumalte.J.T.(1989)N. Engl. J. Med.32. 647-652, I0 Parkos. CA.. Allen. R.A., Cochrane. CG. and Jesa.~hs.AJ. (1987)J.Clin. invest.80. 732-742. !I Nugent,J.H.A., Gralzer,W. and Segal.A.W.(1989)Biochem.J. 264, 921-924. 12 Royer-Pokora.B.. Kunkel.LM., Monaco.A.P. (}off.S.C. Newburger.P.E.,Baehner.R.L, Cole.ES..Cnrnune.J.T.and O~ns. S.H. (1980)Nature322,32-38. I3 Teahan. C, Rowe, P., Parker. P.. Tol~, N. and SegaL A.W. f1987)Nature327, 720-721. 14 Cr,.~. A.R..Jones.O.T.G.,Harper.A.M.and SegaLA.W.(1981) Biechcm.J. 194,599-606. 15 Bellavite, P., Cross, A.R., Serra, M.C, Davoli, A., Jones. O.T.G. and Ro~si. F. (1983) Biechim. B.;ophys.Acla 746. 40-47. 16 lizuka, T., Kanegasaki, S., Makino, R., Tanaka, 1". and lshimura, Y. (1985) J. Biol. C'Tem. , 260, 12049-12053. 17 [.utter, R, van Schaik, M.LJ.. van Zwielen, R., Wever, R., Roos, D. and Hamers, M.N. (1985) J. BIOl.Chem. 260. 2237-2244. 18 Yamaguchi. T., Hayakawa, T., Kaneda. M., Kakinuma. K. and Yoshikawa. A. (1989) J. Biol. Chem. 264. !12-118. 19 Hata-Tanaka, A. Ch~a, T. and Kakinnma. K. (1987) FEBS Lett. 214, 279-284. 39 Kakinuma, IL and Fujii, H. (1989) Prec. Int. Conf, BiOl~hOton,in Advances in Biophoton (lnaba` F., ed.), Vol. 2, Springer-Verlag, Tokyo, in press. 21 Hurst, J.IC, Loehr, T.M., Cumune. J.T. and Rosen, H. (1991) J. Biol. Chem. 266. 1627-1634. 22 Ueno, I , Fnjii, S., Ohya-NishigechioH., lizuka, T. and Kanegasald, S. (1991) FEBS Len. 281, 130-132. 23 Kakinuma, K. (1974) Biochim. Biophys.Acla 348, 76-85. 24 Wakeyama, H., Takeshige, K., Takayanagi, R. and Minakami, S. (1982) Biochem. J. 205, 593-601. 25 Kakinuma, IC, Fukuhara, Y. and Kaneda, M. (1987) £ Biol. Chem. 262, 12316-12322. 26 Agner, IC (1958) Acta Chem. Scan, 12, 89-94. 27 Bolscher, GJ.M., Plat, H. and Wever, R. (1984) P.iochim. BIOphys. Acta 784, 177-186. 28 Fujii, H. and Kakinuma` K. (1991) Biechim. Biophys. Acta 1095, 201-209.
246 29 Cross, /kW., Higson, F.K., Je~.-s, O.T.G., Harper, A.M. and Sesal, A.W. (1982) B~chem. J. 204, 479-485. 30 Ikeda-Sa~to, M. and Pr/ece, tLC. (1985) J. Biol. Chem. 260, 8301-8305. 31 Wever, R., ~ M.N, Wccning, R.S. and Roos, D. (191~)} Fur. J. Baghem. 108, 491--495. 32 WaU~er,F.A_, Reis, D. and Balke, V.L (1984) J. Am. Chem. S¢c. I06, ~ . 33 MmeL F. amJ Vigna~ P. (1984) Btochim. B~ph~. Acta 764, 213-225.
34 t[edc~tedt, L ~ t Anderson, K.K. Uq86) J. Bacteriol. 167, 735-739. 35 Fr~en i-L ~ M.R., Hcdcrstcdt, L, Ander:~o~, K.K. and Thomson+ AJ. (1990) B~chim. B ~ / s . Acta I041, 207-215. 36 Palmer, G. ( I ~ ) l~ochem. Soc. Trans. 13, 548-560. 37 M[Ifi. 11". Fujfi. IL and Kakinuma. IC (199"2) J. Biol. Chen~ in press.
239
© 1992Elsev;erSciencePublishersB.V.All rightsreserved0167-4889/92/$05.00
BBAMCR13226
Electron paramagnetic resonance studies on cytochrorne b-558 and peroxidases of pig blood granulocytes Hirotada Fujii and Katsuko Kakinuma Department o[ Inflammation Research. The Tokyo ~letropolitan bzstituteof Medical Science, Bunkyo-ku. Tokyo (lapan)
(Received 3 April 1992)
Keywords: Granulocyte;Neutrophil;Cytochromeb-558;EPR;NADPHoxid~Lse;(Pig) Low-temperature electron paramagnetic resonance (EPR) spectrometry on granulocytesprepared from pig blood was carried out with concentrated cellular and subccllelar fractions to characterize EPR signa!s of cytochrome b-558 (.cyt b-558). A thick cell suspension (-- 2.10 '~ cells/ml), c~ntaining mostly neutrophils, showed typical high-spin EPR signals due to myeloperoxidase (MPO) and a low spin signal at a g value of around 3.2. A similar thick granulocyte suspension containing eosinophils showed not ,nly these signals but also low spin heme signals at g values of 2.86, 2,13, and 1.66, which have been reported to be of cyt b-558 (Ueno et al. 1991, FEBS Lett. 281, 130-132). MPO and eosinophil peroxidase (EPO) were rcle~. ,~.dfrom the membrane fractions with 50 mM phospha.,ebuffer {pH 7.0) containing I M NaCt, and then were highlyeoncentratetl, in which no cyt b-558 was detected by absorption spectra. The signal at a g value of 2.86 was found only in the EPO fraction, suggesting that this signal is derived from a low-spin form of an EPO-complex, but neither from MPO nor cyt b-558. The O;-forming NADPH oxidase associated in the membranes was solubilized with heptylthioglucosideat 0°C and concentrated up to 45 tLM q,t b-558 with no modification of the heme moiety confirmed by its Of-generating activityand lack of carbon monoxide-bindingcapacity. Cyt b-558 showed an anisotropic signal at a g value of 3.2 _+0.05, which was cyanide-insensitiveand reducible with reductants. The signal intensity was concentration dependent, suggestingthat the g = 3.2 signal is characteristic of the low-spin heme iron in cyt b-558.
Introduction
Granulocytes, such as neutrophils and eosinophils, respond by generating the superoxide anion (Of) in response to invading microorganisms [1]. This response is essentially due to the activation of a membrane-associated NADPH oxidase system [2], which has been hypothesized to be a multi-component electron transport system consisting of a membrane-bound flavoprotein [3-5], b-type offochrome [6,7], and cytosolic components [8,9]. Since the discovery of the absence of b-type cytochromes in the neutrophils of patients with X-linked chronic granulomatous disease (CGD) [6] that cannot produce 0 2 when stimulated, this cytochrome has been extensively studied as a key enzyme in the Oj-generating system of neutrophi!s. This cytoehrome, called cytochrome bs58 (off b-558), was alc,o found in n~noq, tes, eosinophils, and maerophages [11. Cyt b-558 is a heterodimer consisting of a heavily glycosylated ql-kDa subunit (,8-subunit) and a non-glycosylated 22
Correspondenceto: K. Kakinama,Departmentof InflammationResearch. The TokyoMem'~olitanInstituteof MedicalScience,18.22 Honkomagome3-chorea,P,unlqo-ku,Tokyoi13, Japan.
kDa subunit (a-subunit) [10,11]. The genes for both these molecules have been cloned and sequenced [12,13]. Although detailed structural and immunological data on cyt b-558 have been reported [I,q--13], it is not as well characterized functionall~;. Several researchers have suggested that cyt b-558 is the terminal oxidase of the respiratory electron transport chain due to its unusually low redox potential (E,, ~- -240 mV) [14] and carbon monoxide (CO)-binding capacity [14,15]. However, this viewpoint has been challenged by other observations that cellular cyt b-558 does not bind CO [16]. These contradictory results might be due to structural modification of 'he heine; i.e., solubilization or purification of cyt b-558 has been reported to gi,te rise to increased CO-binding affinity and a large shift in its reduction potential [14,15,17,18], Electron paramagnetic resonance (EPR) spectroscopy on off b-558 has been reported by several groups [19-22]. Previously, an immunoprecipitated fraction from solubilized NADPH oxidase showed cyanide-insensitive high-spin signals, but only in the stimulated preparations [19], which had been considered to be due to off b-558 but were later found to be due to modified myeloperoxidase (MPO) [20]. Recent
240 EPR studies of a solubilized fraction from neutrophil membranes containing 11 ,aM cyt b-558 showed no heine signals, except that from high-spin MPO [21]. On the other hand, low-spin heine signals at g values of 2.85, 2.21 and 1.67 were recently found for 03 b-558 in a thick neutrophil suspension [22]. in order m characterize the EPR spectma of cyt b-558, it is critical to prepare a highly concentrated 03 b-558 sample, with little contamination from other hemoproteins, and with no modification of the 03 b-558 heme moiety. In the present work, we first examined the heme proteins, 03 b-558, MPO and eosinophil peroxidas¢ (EPO), at both cellular and subeellular levels, to distinguish their EPR signals. A low spin heine signal detected in concentrated cyt b-558 fractions, probably derived from the heme iron of cyt b-558, is discussed, based on spectrophotometric results obtained for all the cellular and subcellular samples. Materials and methods
Materials The fatty acids, myristic acid and arachidonic acid, were from Wako Pure Chemical, Tokyo, and were dissolved in ethanol as descrl3md previously [23]. Heptylthioglucoside (HTG) was purchased from Dojindo Laboratories, Kumamoto. NADPH and NADH were from Oriental Yeast, Tokyo. 5uperoxide dismutase (SOD), cytochrome c (type VI, from horse heart), and glucose oxidase were purchased from Sigma. Other reagents were of analytical grade.
Preparation of gramdocytes Granulocytes were obtained from pig blood as reported previously [5,24] by the Conray-Ficoli differential density centrifugation method. The erythrocytes contaminated in the huffy coat were hemolyzed with a large volume of ice-cold 0.2% NaCI solution for 30 s, and then the preparations were promptly mixed with an equal volume of ice-cold 1.6% NaCI solution to restore the isotonicity. The resulting cell preparation contained about 90% neutrophils and other cells, in which the percent of eosinophils varied. Cell populations were determined by microscopy. Stimulated and unstimulated cells were prepared with and without myristate, as descn'bed previously [25] and were then collected by centrifugation, mixed with ice-cold 0.34 M sucrose containing 50 mM phosphate buffer (pH 7.4) at 2" l0 s cells/ml and promptly frozen, as described previously [25].
of neutrophils and eosinophils, respectively. For neutrophils, the soaication time was set at 6 x !.5 s at 20 W with intervals of ! s. The sonicated suspension, which contained more intact eosinophils than new trophils, was centrifuged at 500 x g for 5 rain at 0-2°C to remove unbroken cells and nuclei. The post-nuclear supernatant was centrifuged at 100000 x g for 60 rain at 0 - T C to collect membrane vesicles containing the NADPH oxi-dase and azurophil granules, as membrane fraction l. For eosinophils, the sonication time was set at 30 s ( 1 5 x 2 s) with intervals of 1 s and then centrifuged in the san~.e manner to obtain membrane vesicles containing the oxidase, azurophil and eosinophii granules, as membrane fraction II. Azurophil and eosinophil granules were detected spectrophotometrically as described below.
Separation of MPO and EPO from membranefractions Membrane fraction ! (I-10 ~° cell equivalent) obtained by the above procedure was mixed with 100 ml ,,,f 50 mM phosphate buffer containing 1 M NaCI at 0-2°C for ! h, and then centrifuged at 100000 × g for 30 rain at 0-2~C. The resulting supernatant contained fairly large amounts of MPO. The EPO-rich fraction was separated from the Membrane fraction il by the treatment with the same NaG-containing buffer and centrifugation in the same manner. The two supernatants were concentrated with Amicon centricone-30 microconcentrator by centrifugation. Concentrations of MPO and EPO were determined spectrophotometrically using the absorbance coefficients of the Soret bands: 89 cm -I mM at 428 nm for MPO [26] and 110 cm -t mM at 412 nm for EPO [27].
Solubilization of O't b-558 The membrane-associated 03 b-558 was solubilized at if'C, as described previously [28]. The membrane fraction 1 obtained by the above procedure was suspended a~: a final concentration of :~ mg protein per ml in a solubilizing mixture consisting of I% (w/v) heptylthioglucoside (HTG), 30% glycerol and 50 mM phosphate buffer (pH 7.0). The mixture was gently stirred in an ice-bath for 30 rain and then centrifuged at 100000 x g for 60 rain to obtain the solubilized ojt b-558 supematant. The cytochrome rich fraction was concentrated with AmJcon centricone-30 microconcentrators by centrifugation at 5000 x g for 10 h at 0 - 5 ~ , which concentration was dete .rmined from the difference s ~ r a of dithionite reduced minas oxidized samples, using the molar extinction coefficient of 21.6-103 (558 minus 540 rim) [29].
Destruction of neutrophils and eosinophils The frozen cells (1-10 l° cells/50 ml) were thawed and sonicated at {PC by two different methods in a Branson Sonifier (model 185) equipped with a controller to set different sonieation times for destruction
Spectrophotomelric measurements Absorption spectra were measured at 25°C in the range of 400 and 600 or 700 nm in a microcuvette (10 mm light path, 3 mm width) in a Unisoku Biospec-
241 trophotometer US-401 (Unisoku, Osaka), controlled by a computer, NEC PC9801.
Z03
CO-bbuling experiment The reduced minus oxidized difference spectra of cyt b-558, either with NADPH or dithionitc, were measured with and without CO over 400 and 600 nm in a microcuvette (10 turn light path and 3 mm width) containing a reaction mixture consisting of 60 #i of the solubilized sample and an equal volume of 0.I M phosphate buffer (pH 7.0). The concentration of cyt b-558 used was 2 to 3 pM. Strict anaerobic conditions were aeh,~eved according to the reported method [28] using pure argon and a system of glucose and glucose oxidase. For CO difference spectra, a NADPH or dithionite-reduced solubilized sample was equilibrated with o~gen-free CO of purity greater than 99.95% for 15 rain at 4°C. ;
NADPH dependent O2-generating activity was measured p~ the rate of cytochrome c reduction in the absence of SOD, minus the rate with SOD, in a Hitachi 556 spectrophotometer [2].
1 1000
EPR spectra EPR spectra were recorded in a Jeol (JES-FE) X-band EPR spectrometer equipped with LTR HeliTran liquid helium transfer refrigerator (Air Products and Chemicals). The conditions for measurements were as follows: microwave power, 5 roW; modulation amplitude, 6.3 G at 100 KHz; response, 0.3 s; sweep time, 4 min, temperature, 5 to 15 K. For EPR spectrometry of cells, a thick cell sample ((1.8-2.3). 109 cells/hal) was obtained by centrifuging a cell suspension (5. l0 s cells/ml) at 200 × g for 5 rain at 15°C, and was put into an EPR tube (4 mm i.d.). For measurements of EPR spectra of the peroxidases, the MPO or EPO fraction concentrated at 50 to 70 p,M as descn'bed above was transferred into the EPR tu~e, respectively. For the measurement of EPR spectrcmetry of the cyt b-558 fraction, the solubilized cyt b-558 fraction was concentrated and transfe~Ted into the EPR tube at a concentration of 10 m 45 # M cyt b-558. Itemlts and Discassien
EPR spectra of oanulocytes The EPR spectrum of granulocytes (1.8-109 cells/mi) at 10 K is depicted in Fig. 1A, which shows a homogeneous high-spin heine iron spectrum with rhombic symmetry. The g values of 7.03, 5.20 and 1.94 are identical to those of MPO [30]. In addition, low-spin heme signals with g values at 2.86, 2.13 and 1.66 were detected as well as a new unassigned broad signal around g -- 3.2. The EPR spectrum of another sample
151
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Field (gauss) Fig~ !. EPR spectra of pig blood granulocytes.(A) Granulocyles (1.8-109 cells/ml);B: Granulocytes(23.109 ceils/ml);(C) grauulocytcs in (A) treatedwith 2 mMpotassiumcyanide.The EPR instrumentalconditionswereas follows:microwavepower.5 mW;modula. tion,6.3 G at 100kHz;responsetime,0.3 s; temperature.10IL
(2.3.109 cells/ml) in Fig. 1B shows the high-spin EPR signal of MPO but not the low-spin signal at g = 2.86 which appeared in Fig. 1A. The concentrations of cyt b-558 in these two samples were about 16.2 and 20.7 /zM in Fig. 1A and B, respectively. Furthermore, this low-spin signal intensity was not dependent vn the cyt b-558 concentration, suggesting that the low-spin heine signal at g = 2.86 is not due to cyt b-558. When the cells in ~:ig. iA were exposed to 2 mM cyanide, the high-spin MPO signals disappeared and new signals due to the MPO-cyanide complex appeared at g values of 2.84, 2.17 and 1.61 (Fig. IC). The ferric low spin signal at g = 2,86 (Fig. IA) was not affected by the addition of cyanide. The signal at gl = 2.86 was dis. tinct from that of the MPO-cyanlde complex (gt = 2.84), but two other signals, g2 and g3, overlapped those of the MPO-cyanide complex. After considering the cyanide insensitivity and the dithionite reducibility, these signals were previously assigned to cyt b-558 by Ueno et al. [22] with similar cell preparations. However, these granulocyte preparations contained not only neutrophils but also eosinophils, which have different types of granules containing various metaloproteins.
242 Thus, we examined the individual lysos~mal iron pro~eins ( ~ 0 aa~ EPO, ;~n membrane fract~:.-._~ ~ f o r e fmal~ assigning the metalopmtein with g~ -- 2.86.
Absorption spectra and EPR spectra of peroxidase-rich
fracture M P O and E P O releasedwith I M NaCI from the membranes were analyzed by absorption and EPR spectrometries.The differenceabsorptionspectrum of a reduced m/nus oxidized MPO fraction is shown in Fig. 2A. The large absorption peaks at 473 and 640 nm are attn'buted ~ to MPO [26], and while the small peaks at 560 nm and :/95 nm can be aun'butezl mainly to MPO, but also to EPO [31], indicating that the _fr_,~ction ~ Fig. 2A contains predominantly MPO and only a small fraction of EPO. On the other hand, this fraction does not contain cyt b-558, since no absorption peak appeared at 425 nm (Fig. 2A) [6]. The highly concentrated (72 p M ) MPO fraction was subjected to EPR spectroscopy as shown in Fig. 2B. The EPR
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FMd (ga,~) Fi~ 3. Sw.ctmsce~ pmpe~s of the EPO f."-~fion.CA)Difference spectrum of dithim~-reduced n~us oxk!mM state of the EI'O ~ The com~enttationsof EPO and MPO gere 66 ~M and ]6 ~M, n:~ectively. (B) EPR sigch'ma of the EPO haction. (C) EPR speannn of the EI'O ~ treated with 2 mM cyanide recorded at l0 K. The insmnnentai conditions for (13)and (C) ~ere identical to those in ~g. 1.
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Field (gauss) F'~ 2. S p e c t ~ properties of the MPO fraction. (A) Difference ebsorptionspectremof dithionite-reducedminusoxidizedMI'O fraction. The concentration of MPO in the fractionwas 72 pM. (B) EPR spectrum of the oxidized state of the MPO fraction. (C) EPR stpectrum of the MPO fraction treated with 2 mM c/ankle. Instrumental conditio,s for (B) ancl(C}were identical to those in Fig. 1.
spectrum shows high-spin ferric heine signals with rhombic symmetry (g values of 7.02, 5.22 and 1.96), consistent with the previously reported spectrum of MPO [30]. No other EPR signals were detected (Fig. 2B). Addition of 2 mM cyanide to the MPO fraction resulted in the disappearance of the high spin signals with a concomitant appearance of new MPO-cyanide complex signals at g = 2.84, 2224 and 1.66 (Fig. 2 0 . We also examined an EPO-rich fraction (see Materials and Methods). The difference absorption spectrum is shown in Fig. 3A, with typical EPO peaks at 450 rim, 563 nm and 598 nm [31]. The additional band at 640 .,un is due to either EPO or MPO; the large
243 band at 470 nm is due to MPO. Using reported extinction coefficients of EPO [27] and MPO [26], the concentrations of EPO and MPO in this fraction were calculated to be 66 pM and 16 pM, respectively. Again, no cyt b-558 was detected in this highlyconcentrated fraction due to the lack of a peak at 425 nm (Fig. 3A) [6]. The EPR spectrum of the EPO-rich fraction showed both high-spin and low-spin ferric heine signals with g values of 6.86, 5.23 and 1.96, and 2.87, 2.21 and 1.67, respectively.These high-spin heme signals were close to those reported previously with cellular and purified EPO, g = 6.60 [31] and 6.50 [27], respectively.The low-spin heine signals were similar to those for the cells shown in Fig. 1A, Figure 3C shows EPR spectrum of the EPO-rich fTaction treated with 2 mM cyanide. The ferric high-spin heine signals at g = 6.86 and 5.23 (~g. 3B) decreased markedly; instead, the low-spin berne signals from the EPO- and MPO. cyanide complexes appeared at g values of 2.~;3, 2.21 and 1.65. On the other hand, the low-spin signal at g = 2.87 (Fig. 3B) was cyanide-insensitive.Redu.aion of the EPO-rich fraction with sodium dithionite resulted in the disappeazance of both high-spin (g = 6.86 and 5.23) and knv-spin (g = 2.87) heme signals (data not shown). The EPR signal at g = 186-187 (Fig. IA and Fig. 313) has been tentatively assigned to cyt b.558 by Ueno et al. [22]. However, we found that this low-spin ~gnal at g = 186 was sim~ar to that in the cyt b-558free and EPO-rich ~ (Fig. 3B). Therefore this g = 2.86 signal is not from cyt b-558, but is likely due to a low-spin EPO-complex,which might be formed from high-spin EPO with an endogenous fig,and(s)such as OH- and (21- [30]. Furthermore, Ueno et al. [22] obtained a tetragnnal ct~ml field spfitting of 4.96, and thought it to be too large for that of a b-type cytochrome, 3.11-4.25 [32]. However, the value of 4.96 is well within the range calculated for low-spincomplexes of EPO, MPO and catalase (3.66-5.25) [27].
Absorption and EPR spectra of solubilized cyt b-558 Treatment of membrane vesicles with 1% HTG resulted in a high yield of so!ubllizatiou of both cyt b-558 (98.6 + 4.9%, n -- 6) and NADPH oxidase (94.3 + 5.4%, n = 6) in contrast to very slight solub'dization of MPO [28]. Fig. 4 shows difference absorption spectra of dithionite-reduced minus oxidized membrane vesicles (Fig. 4A) and the solubilizcd oxidasc fraction (Fig. 4B), respectively. The peak at 470 nm due to MPO detected in the difference spectrum of membrane vesicles (A) was totally absent in the soinbllized fraction (B), confirming that the solubllized fraction contains negligibleamounts of MPO. On the contrary, the HTG-solnbilizedfraction contained a highly active NADPH oxidase, which produced 250-475 nmol 021nfin per mg of protein (n =6), from my~'istatestimulated cell membraneg while showing negligible
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650
W~velengLh (rim) Fig. 4. Dithionite-reduced minus oxidized difference spectra of membrane vesicles (A) a,:d the solebilizcd ~.~ b-558 fraction (B). Membra,e vesicles (5 mg protein/m|) and solubilized cyl b-558 fraction (3 mg protein/ml) were prepared in 0.1 M p,hosphate buffer (pH 7.0),
activity with unstimulated cell memb~nes, yet both HTG-solubilized fractions contained almost equal amounts of cyt b-558, 317-333 pmol/mg of protein (n ; 6) [28]. While cyt b-558 hardly binds CO in sit~ [16], the solubilization and isolation from its hydrophobic environment may have resulted in a change in the environment of the heine of cyt 6-558, partly tee,yard .~omething like high-spin ferric heine, consequently increasing its CO-binding capacity, as reported previou~.qy from this laboratory [18].Therefore, wc carefullyexamined the CO-binding capaci~ of HTG-solubilized cyt 6-558. The heme of cyt 6-558 in ~he solubilizedfraction was reduced with 0.1 mM NADPH under anaerobic conditions by incubation for 20 rain [28], and then the reduced minus oxidized difference spectrum wa~ obtained (Fig. 5A). After equilibrating the reduced sample with CO for 15 rain, the difference spectrum was measured (Fig. 5A), Both spectra showed no difference in the a and p bands before and after mixingwith CO. Fig. 5B shows the Co-dlfference spectrum of reduced cyt b-558 fraction in the absence and presence of CO, giving evidence that reduced cyt b-558 in the presence of the physiologicalsubstrate, NADPH, does not bind CO; that is, cyt b-558 may not form an oxygenated complex, consistent with the evidence by lizuka et al. [16]. For comparison, the difference spectra of dithionRe-reduced minus oxidized cyt b-558 were measured
244
15 rain aiIer the incubation with and without CO. The spectra obtained showed that a small portion of reduced cyt b-558 (about 10%) complexed with CO in the dithionite-redueed state (Fig. 5C). In fact, it may he poss~le that dithioni~e might induce this structural modification of the cyt b-558 heine. Overall these r ~ B indicate that the CO-binding capacity of HTGsolubilized cyt b-558 is poor, its binding rate is very slow [33], and the cnordination environment of the solubilized cyt b-558 heine iron with HTG is not modified, as compared with that in cells. The HTG-solobilized sample from either stimulated or resting cell membranes was concentrated to 45 #M cyt b-558. The EPR spectrum is shown in Fig. 6A.
A = 0.051
4Zl 7.03
5.23 e l
320
fl
.•
!
3-20
2.23
- CO i
iO00
-
~
-
2000
~
3000
4000
Field (Eauss) l~y. 6. EPR %oec~raof con~nlrated cyt/7-558fractions from unstimulat~! celk measured at 10 K, (A) EPR spectrum of the highly concentrated fractioncontaining45 p M cyt b-558.(B) 2 raM cyanide was axkled to the cB b-558 baction of (A). (C) Reduction of the cyt =CO
B
r.
5
xl0
o ....
=CO
400 r " " " 4 ~ ; . . . . . . 500 . . . .
8oo
x 10
~50""" 600
Wavelength (nm) F*g. 5. Difference absorp~oo spec~ of NADPH-rcduced minus mid~ed state of t~e c!a b-558 fraction in the absence and presence of CO (A, +_CO)and CO-difference spectra of the cyt/7-558fraction reduced with NADPH (9) and dithionite (C). (A) The ojt b-558 fiactioD u)lubilized from stimulated cells in 0,1 M phosphate buffer (pH 7.0) was mixed with an I mM NADPH (final concentration 0.1 raM)anaerobicalb'for 20 rain.The ~ncentmfionof the cyl b-558in
the fractionwas 2 t~M..Afterthe samplewith NADPHwas equilibrated with o'~gen-freeCO (> 99.95%) for 15 min at 4~C, the differencespectrmnwas measured. (B) The CO-differenceb ' ~ m was calculatedfrom the red~'ed spectrawith N A D P H in the presence and absence of CO. (C) The CO~ifferencespectrum was
calculatedfromthe reduced~ecaa withdithionitcin the presence and absenceof CO. The ~ of reducedc~/7-558withdithionite wereraeasured15rainafterthe incubationwithand withoutCO.
/>-558fraction in (A) with dithionite. The instrmnental conditions for (A), (B) and (C) were identicalto those in Fig. I.
Several signals were detected at g values of 7.03, 6.03, 5~3 and 4.23 in the low field region. The g values, 7.~: and 523, were identical to those reported for MPO in the ferric high-spin state [30]. The signal at 4.23 resembles that for non-heine high-spin ferric species. In the high-field region, a distinct EPR signal at a g value of 3.2 + 0.05 was observed. In addition, a small signal was also detected at a g value of about 3.0. Other minor signals could correspond to coppercontaining compounds (g -- 2.08) and o;ganic free radicab (g = ZOO).The EPR spectrum of a similar sample, prepared from stiwulated membranes, was essentially the same as that in Fig. 6A (data not shown). Note that in Fig. 6A, there was no signal at g-- 2.86-2.87, again giving evidence that a g = 2.86-2.87 signal is not due to cyt b-558. Figure 6B shows the EPR spectrum of the cyt b-558 fraction treated with 2 mM cyanide. The ferric high-spin heine signals (Fig. 6A) at g -- 7.03 and 5.23 disappear¢~ while the low-spin heine signals for MPO-cyanide complex appeared at g values of 2.84 and 223, respectively. An unassigned high-spin signal (g -- 6.03) from Fig, 6A also decreased markedly upon cyanide addition. Thus, the high-spin signals at g--
245 7.03, 6.03 and 5.23 were not due to cyt b-558. The low-spin ferric signals at g values of 3.2 and 3.0 did not disappear in either 2 mM cyanide (Fig. 6B) or 4 mM azide (data not shown), yet completely disappeared in dithionite, as shown in Fig. 6C. When the concentrated cyt b-558 fraction was reduced with NADPH under anaerobic conditions, the EPR signals at g = 3.2 and 3.0 diminished (data not shown). The EPR signal ~.ntensityat a g value of 3.2 increased with increasing ey'~ b-558 concentration, but the signal intensity at around g = 3.0 varied in different samples. T h ~ results suggest that the g = 3.2 signal is due mainly to cyt b-558. The other EPR signal at g = 3.0 has not been assigned yet but ~i.~ht ~ due to different state of cyt b-558. The axial amino acid liga::ds to the berne of cyt b-558 have not been identified, but Hurs~ et al. [21] recently reported from resonance raman spectra that the heine is a low-spin six-coordinate with imidazole or imidazolate axial ligation. The g:values of b-type cytochromes are reported to change with the axial ligand; i.e., thiolate-imidazole iigation (g = 2.34-2.46), aliphatic amine-imidazole (g=3.33-3Al), and bis-aliphatic amines (g = 3.57-3.63) [32]. The g = 3.2 observed in this study seems higher than those reported for most b-type cytochromes with bis-histidine coordination, 2.69-3.03 [32]; however, recently, several membranebound b-t3~e cylochromes have been reported to give g values higher than 3.1 [34, 35]. Friden et al. [35] found that Bac/lhts subt///s cytochrome b-558 was a low-spin hemoprotein with a highly anisotropic EPR signal, g = 3.5, and that this cytochrome b-558 was a bis-histidine coordinated hemoprotein, using low teml~erature EPR and magnetic circular dichroism. Thus, the present results, with a highly anisotropic signal at g = 3.~ may be compat~e with b~histidine coordination of neutrophil cyt b-558, as predicted by its resonance raman spectroscopic results [21]. The othcr known g values (e.g., g2 and g3 ) for cyt b-558 were not observed in Fig. 6A, due possibly to overlapping by other signals. Palmer [36] has proposed that if the two imidazole-ring planes of the ligating histidines are gradually rotated from a parallel orientation (g~ = 3.0) toward a perpendicular orientation, the g~ value will increase, while the g2 and g3 components will become progressively smaller. Such a perpendicular orientatio,) of the ligating histidine groups might exist in neutrophil ~ t b-558, resulting in smaller g2 arid g3 components of its EPR spectra. In conclusion, all of these results suggest that the signal at a g value of 33.± 0.05 is derived from cyt b-55g, characteristic for a hexa-coordinated low-spin heme protein, in accord with its CO-insensitivity. Recently, this assignment has been further confirmed from EPR spectra of purified cyt b-558 [37].
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