©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Electron Spin Resonance Studies on Neutrophil Cytochrome b
EVIDENCE THAT LOW-SPIN HEME IRON IS ESSENTIAL FOR O GENERATING ACTIVITY (*)

Hirotada Fujii (1)(§), Michael K. Johnson (2), Michael G. Finnegan (2), Toshiaki Miki (1), Lucia S. Yoshida (1), Katsuko Kakinuma (1)

From the (1) Department of Inflammation Research, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 18-22, Hon-komagome 3-chome, Bunkyo-ku, Tokyo 113, Japan and the (2) Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cytochrome b purified from pig neutrophils was studied to characterize the spin state of the heme iron in relation to its O generating activity. ESR spectra of cytochrome b either from resting or stimulated neutrophils showed a low-spin hemoprotein with g of 3.2, 2.1, and 1.3 (estimated). At physiological pH, the oxidized cytochrome b is in a purely low-spin state. On lowering or raising pH from 7, the spin state changes to high-spin. The ESR spectrum of high-spin cytochrome b was identical to that of methemoglobin, suggesting that the axial-ligand type in both hemoproteins may be the same, i.e. histidine is the fifth ligand. The ratio of the low-spin to high-spin heme in cytochrome b was evaluated by magnetic circular dichroism spectroscopy. The pH of cytochrome b was varied to form different ratios of the low-spin to high-spin states of the heme, and its O generating activity was examined in cell-free systems. O forming activity decreased concomitant with loss of the low-spin heme, which provides direct evidence that the low-spin state of cytochrome b is essential to generate O and the heme retains the low-spin state through the redox cycle.


INTRODUCTION

A phagocyte-specific cytochrome b is an essential component of the membrane-bound NADPH oxidase system which produces superoxide anion (O) in response to the invading microorganisms (1, 2) . Cytochrome b is a heterodimer consisting of large and small subunits, both of which have been sequenced (3, 4) . The large subunit of cytochrome b is postulated to be a membrane-bound flavocytochrome from its amino acid sequence homology with other flavoproteins (5, 6, 7) , affinity labeling with NADPH analogues (6, 8) , and binding of FAD (9) , but other flavoproteins, such as 77-kDa protein (10) and nitro blue tetrazolium reductase (11) , were also reported as candidates for NADPH dehydrogenases. In spite of the flavocytochrome hypothesis, no one has isolated an FAD-containing cytochrome b from neutrophil membranes. Cytochrome b has been postulated to function as the terminal oxidase of the respiratory electron transfer chain due to its unusually low redox potential (E = -245 mV) (12) , CO-binding capability (12, 13, 14) , and rapid reoxidation rate (15, 16) . We carefully examined the CO-binding capacity of cytochrome b using solubilized membrane fractions from either resting or stimulated cells, and confirmed that the CO-binding capacity of undenaturated cytochrome b is very poor (17) . Furthermore, NADPH-dependent reduction of cytochrome b in the stimulated state of the oxidase is as slow as that in the resting state, which may not explain the O generating activity of the NADPH oxidase system (18, 19) . Recently, we purified cytochrome b without losing its O forming activity in a cell-free system (11) and succeeded in assigning the ESR signal at g value of 3.2 to the hexa-coordinated low-spin heme iron of cytochrome b(17, 20) .

In the present study, we have further investigated the low temperature ESR spectrum of cytochrome b, which was purified and concentrated without denaturation from resting and stimulated neutrophils. The results confirm that both preparations of cytochrome b exhibit a rhombic ESR spectrum with g values of 3.2, 2.1, and 1.3, similar to that of mitochondrial cytochrome b(21) . In addition, the spin state of the heme iron was modified by varying the pH of cytochrome b preparations to yield samples with perturbed high-spin to low-spin ratios. Using these preparations, we studied the correlation of the O generating activity with spin state of these cytochrome b in order to clarify whether the low-spin state of cytochrome b is essential to O generation in the NADPH oxidase system.


EXPERIMENTAL PROCEDURES

Materials

DEAE-Sepharose CL-6B and heparin-Sepharose 6B were obtained from Pharmacia LKB Biotechnology Inc. n-Heptyl--thioglucoside and EGTA were purchased from Dojindo Laboratories, Kumamoto. NADPH was purchased from Oriental Yeast, Tokyo. Sodium myristate and diisopropyl fluorophosphate were from Wako Pure Chemicals Co., Tokyo. Superoxide dismutase, cytochrome c (type VI, from horse heart), and human hemoglobin were purchased from Sigma. GTPS() was a product of Boehringer Mannheim. Other chemical reagents were of analytical grade.

Purification of Cytochrome bfrom Pig Blood Neutrophils

Resting or stimulated neutrophils were obtained from pig blood as described previously (22) and treated with 2 mM diisopropyl fluorophosphate for 20 min at 0 °C. The membrane fraction was obtained from the sonicated neutrophils, and then cytochrome b was solubilized from the membrane fraction with n-heptyl--thioglucoside according to the previous method (18). Cytochrome b was purified from solubilized membranes according to the method reported previously (11, 20) , except that a heparin-Sepharose column was equilibrated with a buffer composed of 50 mM phosphate buffer (pH 7.0), 50 mM NaCl, 10% glycerol, and 0.6% n-heptyl--thioglucoside.

Assay of Cell-free O Production

The O generating activity of the NADPH oxidase was determined at 25 °C in a reconstituted cell-free assay system according to a reported method (11) with slight modification. The pH of the purified cytochrome b preparation was adjusted to a desired pH by mixing with small amounts of 0.5 M NaOH or 0.5 M HCl, and the preparations were kept for 10 min on ice. Then, the pH of such a preparation was readjusted to 7.0 by HCl or NaOH. This cytochrome b preparation was reconstituted by incubating with an appropriate amount of the nitro blue tetrazolium reductase fraction (11) for 30 min on ice. Thereafter, the mixture containing 2 pmol of cytochrome b was incubated for 5 min at 25 °C with cytosol from resting cells, 20 µM GTPS, and an appropriate amount of sodium myristate in the assay medium composed of 50 mM phosphate buffer (pH 7.0), 2 mM NaN, 1 mM EGTA, and 1 mM MgCl, and then the assay medium containing 50 µM cytochrome c was added to the above mixture to give a final volume 0.8 ml. O production was measured with a Hitachi dual-wavelength spectrophotometer (model 556) by recording the reduction of cytochrome c at 550 nm after addition of 125 µM NADPH.

Spectrophotometric Measurements

Absorption spectra were measured at 25 °C in the range of 400 and 600 nm in a microcuvette (10-mm light path, 3-mm width) in a Unisoku Biospectrophotometer US-401 (Unisoku Co. Ltd., Osaka), controlled by the computer, NEC PC9801. The volume of the sample used in the microcuvette was 120-150 µl. MCD spectra were recorded using JASCO J-500 spectropolarimeter interfaced to a Jasco MCD-1B electromagnet. Spectra were recorded in the wavelength range from 300 to 800 nm, at a magnetic field of 1 tesla and at a temperature of 25 °C.

ESR Spectra

ESR spectra were recorded in a Jeol (JES-FE) X-band ESR spectrometer equipped with LTR Heli-Tran liquid helium transfer refrigerator (Air Products and Chemicals Inc.). The conditions for measurements were as follows: microwave power, 0.16-10 mW; modulation amplitude, 10 G at 100 KHz; response, 0.3 s; sweep time, 4 min; temperature, 5 to 20 K. Copper EDTA in water was employed as a concentration standard. For the measurement of ESR spectrometry, both cytochrome b-rich membranes and the purified cytochrome b were concentrated with Amicon Centricon-30 at 5 °C. The pH of the concentrated sample was adjusted by aliquots of 0.5 M NaOH or 0.5 M HCl at 0 °C, and was measured by Cosmo pH Boy-C1 at 25 °C.


RESULTS

Heme Environment of Oxidized Cytochrome b

Cytochrome b was purified from pig blood neutrophils without any denaturation of the heme environment, since the cell-free system employing the purified cytochrome b exhibited high O generating activity. The catalytic activity of the cell-free systems with the purified cytochrome b was 55-63 mol of O produced per s/mol of cytochrome b, which was more than 80% of that of the solubilized membranes, 73 mol of O. The ESR spectrum of the purified cytochrome b prepared from resting neutrophils was measured at 5-20 K, in which a spectrum recorded at 10 K was shown in Fig. 1. No difference in the spectrum was found in purified cytochrome b prepared from the stimulated neutrophils. Cytochrome b in the oxidized state at pH 7.0 showed a low-spin ESR spectrum with g value of 3.2 in accord with that of the previous reports (17, 20) , but the present preparation showed much weaker ESR signals from unknown origins at g values of 6, 4.3, and 2, as compared with the previous signals (20) . In the previous report (20) , only the low field component at g = 3.2 was assigned. The improved ESR data, shown in Fig. 1, clearly show the associated derivative component at g 2.1. On the basis of these two g values, the high field g value is expected near g = 1.3. However, in common with other hemes with similar g value anisotropy, this feature is broad and difficult to discern above the background. The g value anisotropy is not as large as that encountered for mitochondrial cytochrome b exhibiting highly anisotropic low spin-type signals (g = 3.4-3.8), but in common with these species the low-spin resonance of cytochrome b is fast relaxing and requires low temperature (5-20 K) and high microwave powers (10-20 mW) to facilitate observation.


Figure 1: ESR spectrum of cytochrome b recorded at 10 K. The purified cytochrome b was concentrated to 103 µM and the pH in the medium was adjusted to 7.0 with 50 mM phosphate buffer. The ESR instrumental settings were as follows: microwave frequency, 9.04 GHz; microwave power, 10 mW; modulation amplitude, 10 gauss; response time, 0.3 s.



The effect of pH on the absorption spectra of purified cytochrome b was studied at 25 °C, and the series of dithionite-reduced minus oxidized difference spectra measured at different pH are illustrated in Fig. 2. Difference spectra at around physiological pH, having a -band at 428 nm, a -band at 530 nm, and an -band at 558 nm, are characteristic of the low-spin heme. On lowering pH from 7 to 3.1, however, the difference spectra were broadened with concomitant loss of the signal intensities at the -, -, and -bands. Below around pH 4, the -peak was remarkably broadened, followed by the increase in the shoulder at around 437 nm. In contrast to the acidic region, the remarkable change in difference spectra was not seen in alkaline medium until around pH 11, but a slight loss of the signal intensities at the -, -, and -bands was detected. The absorption spectra in Fig. 2 indicate that the pH in the cytochrome b preparation induces a change in the spin state of the heme in both acidic and alkaline medium.


Figure 2: Effect of pH on the reduced minus oxidized absorption difference spectra of cytochrome b at 25 °C. The pH in the samples were (from top to bottom): pH 11.6, 10.3, 9.2, 7.0, 5.0, 4.1, and 3.1. Purified cytochrome b was diluted with 50 mM phosphate buffer (pH 7.0) containing 0.6% n-heptyl--thioglucoside to a final concentration of 2.1 µM. The pH in the cytochrome b preparation was adjusted by adding aliquots of either 0.5 M NaOH or 0.5 M HCl.



The effect of pH on the heme environment of cytochrome b was studied using ESR spectrometry. ESR spectra of oxidized cytochrome b in the pH region from 4.1 to 11.8 are shown in Fig. 3. The ESR spectrum at pH 7 is dominated by the g = 3.2 feature of the low-spin hemoprotein (Fig. 3). On lowering or raising the pH from 7, new ESR signals appeared at g = 6.0 under acidic pH and at a g value of 6.2 under alkaline pH. Both signals are typical of high-spin ferric hemes, and their appearance occurs concomitant with the disappearance of the low-spin signal at g = 3.2. The change in ESR spectra in either acidic or alkaline pH was irreversible. The spin-state transition of the heme from low-spin to high-spin was more pronounced to acidic pH values. At alkaline pH, a new low-spin ESR signal appears, g = 2.42, 2.23, and 1.91, in addition to the high-spin signal at g = 6.2. This most likely reflects an equilibrium between a five-coordinate high-spin heme and a six-coordinate low-spin heme with altered axial ligation. Fig. 4, A and B, illustrates the comparison of the ESR spectrum of cytochrome b at pH 3.5 with that of methemoglobin at pH 7.0. As shown in Fig. 4A, at acidic pH all of the low-spin heme signals at g = 3.2 disappeared and the low-spin heme has been completely converted to a S = 5/2 high-spin heme with g = 6.0 and g = 2.0. The ESR spectrum of methemoglobin at pH 7, a typical high-spin heme, is shown in Fig. 4B, and is identical with that of the high-spin cytochrome b at pH 3.5 (Fig. 4A). This suggests that the heme environment of cytochrome b in the high-spin state is remarkably similar to that of hemoglobin, which in turn implicates histidine as the fifth ligand. The binding of cyanide to the high-spin heme iron in cytochrome b was confirmed by ESR spectroscopy (data not shown), and the CO binding was also confirmed by reduced-minus-oxidized difference spectra, showing shift in its -band from 428 to 420 nm upon binding CO.


Figure 3: Effect of the pH on the ESR spectra of purified cytochrome b. The purified cytochrome b was concentrated to be 85 µM, and then the pH was adjusted as in Fig. 1. The pH values are as follows (from top to bottom): pH 11.8, 10.2, 7.0, 5.0, and 4.1. The instrumental conditions for all spectra were identical to those in Fig. 1, except that the microwave power was set as 1 mW. Receiver gains were scaled as indicated on the figure.




Figure 4: Comparison of ESR spectrum of the high-spin cytochrome b (A) with that of hemoglobin (B). The purified cytochrome b was concentrated to 95 µM, and the pH value in the preparation was adjusted to 3.5, as described in the legend to Fig. 3. Methemoglobin was dissolved into 50 mM phosphate buffer (pH 7). The concentration of methemoglobin used for ESR measurements was 125 µM. The instrumental conditions for all spectra were identical to those in Fig. 1, except microwave power = 0.16 mW.



The ESR signal associated with the low-spin ferric heme in cytochrome b, see Fig. 1, is too broad for accurate spin quantitation. Moreover it is overlapped by other ill-defined signals particularly in the g = 2 region. In contrast, as shown in Fig. 4, the high-spin ESR signal that appears at acidic pH is much more intense and is amenable to spin quantitation by double integration versus a Cu standard under non-saturating conditions. At 10 K, this procedure indicated a spin quantitation of 88 µM. However, this will be an underestimate since the g = 6.0 and g = 2.0 resonance originates from the lowest (M = ±1/2) doublet ground state manifold and no attempt was made to allow for population of M = ±3/2 or ± 5/2 doublets. Nevertheless this is close to the value of 95 µM which was estimated by optical spectrometry based on a reduced-minus-oxidized extinction coefficient (558-540 nm) of 21.6 10 liter mol for cytochrome b(23) . Hence the low pH high-spin ESR signal appears to account for all of heme.

The ratio of the high-spin state to the low-spin state of the heme in cytochrome b was estimated by MCD spectrometry. The MCD spectra in the region 300-800 nm were recorded at 25 °C of purified cytochrome b in the oxidized state as a function of pH. At room temperature the peak-to-trough Soret band MCD intensity of low-spin ferric hemes is at least 20 times greater than that of high-spin ferric hemes (24) . Hence the percentage of low-spin heme as a function of pH can be conveniently assessed based on the peak-to-trough Soret band intensity. On the basis of the ESR data, the pH 7 data point was taken as 100%. Percent of the low-spin heme in cytochrome b obtained by MCD spectrometry is plotted against pH in Fig. 5.


Figure 5: Effect of pH on percent of the low-spin state of the heme () in cytochrome b and O generating activity () of NADPH oxidase system. pH in purified cytochrome b was adjusted to form various different ratios of the low-spin to high-spin heme. The MCD spectra in the region 300-800 nm were recorded at 25 °C of these purified cytochrome b in the oxidized state. Percent of the low-spin state of the heme in cytochrome b was calculated by the intensity of the Soret band MCD. The O generating activity of the purified cytochrome b with different ratios of the low-spin to high-spin heme was measured in a reconstituted cell-free system.



O-generating Activity and the Spin State

To clarify the relationship between the spin state of the heme in cytochrome b and the O generating activity of the NADPH oxidase system, the O generating activity was measured in a reconstituted cell-free system consisting of the purified cytochrome b with different ratios of the low-spin to high-spin heme prepared by changing the pH, as in Fig. 3 . Fig. 5shows the effect of the functional spin state of the heme in cytochrome b on the O generating activity of the NADPH oxidase system. The optimum O generating activity was found in the vicinity of pH 7, which was the same as that observed previously in the stimulated membrane system (25) . On lowering pH in cytochrome b, the O generating activity of the NADPH oxidase system decreased concomitant with the decrease in percent of the low-spin heme in cytochrome b. These results indicate that cytochrome b is in the low-spin state when the NADPH oxidase system produces O in the cell-free system. In order to further confirm that cytochrome b is in the low-spin state when the oxidase system is generating O, ESR spectra of cytochrome b-rich membranes solubilized both from resting and stimulated neutrophils were measured in the presence of NADPH. The spectra are shown in Fig. 6, and indicate that cytochrome b in both preparations is predominantly in the low-spin state. These results provide evidence that the low-spin state of the heme in cytochrome b is essential to generate O in the activated state of the NADPH oxidase system.


Figure 6: The ESR spectra of cytochrome b-rich membranes prepared from stimulated (A) and resting (B) neutrophils. Cytochrome b-rich membranes were prepared by concentrating the NADPH oxidase obtained both from stimulated and resting neutrophils. The concentrations of the heme in both preparations were 48 µM. After incubating cytochrome b-rich membranes with 200 µM NADPH in 50 mM phosphate buffer (pH 7.0) at 25 °C for 1 min, the mixture in the ESR tube was rapidly frozen.




DISCUSSION

Cytochrome b was purified from pig blood neutrophils without any denaturation of the heme environment, since the purified cytochrome b exhibited high O generating activity in the cell-free system. These purified preparations had greatly decreased resonances at g = 6.0, 4.3, and 2.0, compared with earlier reports (17, 20) . The purified cytochrome b prepared in this study exhibited the rhombic ESR spectrum with g = 3.2, 2.1, and 1.3 (estimated), in which the g component is much broader and less pronounced than the g component. Biochemically important hemoproteins found in several electron-transport chains, such as cytochrome b and cytochrome b in mitochondria (26, 27) , exhibit a low field g value which is substantially larger than 3.1, and other g values, g and g, are not readily observed, since the g signal is much broader and less intense than the g signal. From the ESR spectrum in Fig. 1, the heme environment of neutrophil cytochrome b seems to resemble that of bis-histidine b-type hemoproteins found in the mitochondrial electron-transport chain (21, 27, 28) . Low temperature near-IR MCD studies will provide a more definitive assessment of the axial ligation and such studies are in progress.

Under physiological conditions, the purified cytochrome b is in the low-spin state, but at acidic pH the low-spin heme transformed to the high-spin heme, as evidenced by the characteristic high-spin ESR signal at g = 6. The ESR signal at g = 6, which appeared at the acidic pH, was previously thought to originate from other hemoproteins such as hemoglobin contaminated into the purified cytochrome b(20) , but the present study indicates it to be due to a high-spin heme from denaturated cytochrome b. On the other hand, at alkaline pH the low-spin heme is converted to a mixture of high-spin heme and a different low-spin heme with g = 2.42, 2.23, and 1.91. This low-spin ESR signal is very similar to that of cytochrome P450 (29, 30), and a similar signal is also observed in pyridine-treated cytochrome b(31) . This suggests that alkaline pH induces a structural modification in the heme environment of cytochrome b by shifting the fifth heme ligand, histidine, to a nearby thiolate group of cysteine residue. The sixth ligand is presumably hydroxide. This low-spin form was unable to support O generation in the NADPH oxidase system (31) . An alternative candidate for this ESR signal is a low-spin hydroxide adduct with histidyl and hydroxide axial ligation. While we cannot rule out this explanation, the g value anisotropy is more indicative of a species with cysteinate and hydroxide axial ligation.

One of the important characteristics of low-spin hemoproteins that exhibit highly anisotropic low spin-type ESR signals with a low field component g 3.2 is that the ESR signal intensity or the transition probability at the g signal is very small relative to low-spin hemes with g between 2.4 and 2.9 (26) . Hurst et al.(32) failed to observe the ESR signal of cytochrome b in solubilized neutrophil membranes, although their instrument has the sensitivity to detect ESR signals of a typical low-spin hemoprotein, cytochrome c, when examined at similar concentrations. These authors explained the absence of a low-spin ESR signal of cytochrome b by the relaxation-induced signal broadening due to a strong heme-heme interaction. In light of the present work, such explanations are unnecessary and it would appear that cytochrome b contains a magnetically-isolated b-type cytochrome.

All the hemoproteins involved in activation of oxygen, such as cytochrome c oxidase and cytochrome P450, have a high-spin heme, in which the sixth coordination site is open (or weakly coordinated by water) for binding oxygen. In contrast, neutrophil cytochrome b has been shown to contain a low-spin six-coordinate hemoprotein from ESR and spectrophotometric studies (15, 16, 17, 18, 19, 20, 23, 32) . By correlating O production with the percent of low-spin cytochrome b in samples of varying pH, we have shown that the heme is low-spin during the production of O. Furthermore, transient high-spin heme could not be detected, when cytochrome b-rich membranes prepared from stimulated neutrophils were incubated with NADPH. Both results provide evidence that cytochrome b is in the low-spin state while the oxidase system is forming O. Cytochrome b was originally postulated to be the terminal enzyme of the NADPH oxidase system from its CO-binding capability (12) and unusual low redox potential (12) . Yamaguchi et al.(14) reported that purified cytochrome b had a capability to bind CO. However, only high-spin heme iron of cytochrome b, i.e. denaturated form of the heme iron, was found to be capable of binding both CO and cyanide. On the other hand, Isogai et al.(16) reported that the ferrous cytochrome b was rapidly oxidized by O and concluded that an electron is directly transferred from the ferrous heme to O without ligation of O to the heme iron. If O is generated directly from the reduced heme in cytochrome b, some electron acceptors, such as menadione and quinone, might inhibit the electron flow to O. However, these electron acceptors showed no effect on the O generating activity, indicating that all the electrons from NADPH were selectively transferred to O(18, 33) . More detailed studies on the mechanisms of the electron transfer reactions in hexa-coordinated low-spin hemoproteins are clearly called for. The alternative possibility that O is not produced directly from the heme in cytochrome b also has to be considered.

In conclusion, the results present that the low-spin state of the heme in cytochrome b is essential to generate O in the NADPH oxidase system, and that any transient high-spin heme in cytochrome b does not contribute to the O generating activity.


FOOTNOTES

*
This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan. MCD studies were supported by National Institute of Health Grant GM51962 (to M. K. J.). Preliminary account of this work was presented at the International Symposium on Magnetic Resonance in Biomedical Research, November 15-17, 1994, Okazaki, Japan. 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, Honkomagome 3-chome, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3823-2101; Fax: 81-3-3823-2965; E-mail: fujii@rinshoken.or.jp.

The abbreviations used are: GTPS, guanosine 5`-(-thio)triphosphate; MCD, magnetic circular dichroism spectroscopy; mW, milliwatt.


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