©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mechanism of Electron Donation to Molecular Oxygen by Phagocytic Cytochrome b(*)

(Received for publication, December 29, 1994; and in revised form, January 26, 1995)

Yasuhiro Isogai (§) Tetsutaro Iizuka Yoshitsugu Shiro

From the Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Phagocytic cytochrome b is a unique heme-containing enzyme, which catalyzes one electron reduction of molecular oxygen to produce a superoxide anion with a six-coordinated heme iron. To clarify the mechanism of the superoxide production, we have analyzed oxidation-reduction kinetics of cytochrome b purified from porcine neutrophils by stopped-flow and rapid-scanning spectroscopy. Reduced cytochrome b was rapidly reoxidized by O(2) showing spectral changes with clear isosbestic points, which were also observed during the reduction of ferric cytochrome b with Na(2)S(2)O(4) under anaerobic conditions. The single turnover rate for the reaction with O(2) linearly depended on the O(2) concentration but was not affected by addition of CO. The rate of the reaction decreased with an increase of pH giving a pK of 9.7. Under complete anaerobic conditions, ferrous cytochrome b was oxidized by ferricyanide at a rate faster than by O(2). The thermodynamic analysis shows that the enthalpic energy barriers for the reactions of cytochrome b are significantly lower when compared to the autoxidation of native and modified myoglobins through the formation of the iron-O(2) complex. These findings are most consistent with the electron transfer from the heme to O(2) by an outer-sphere mechanism.


INTRODUCTION

Molecular oxygen is crucial for oxidative phosphorylation and other important chemical reactions in living cells. In many processes of the metabolism, O(2) is transformed to reduced or activated forms by heme-containing enzymes. Phagocytic cells such as neutrophils and macrophages also use O(2) to produce activated oxygen species, including hypochlorous acid and hydroxyl radicals, for killing microorganisms during phagocytosis. The consumption of O(2), called ``respiratory burst,'' is accompanied by oxidation of NADPH and production of superoxide anions (O(2)) (1, 2, 3, 4, 5) . The O(2)-producing reaction is catalyzed by a membrane-bound electron-transferring enzyme system, ``NADPH oxidase,'' in which cytochrome b is the only identified component that contains redox centers(6, 7, 8, 9, 10, 11, 12) . In previous studies(13, 14) , we have demonstrated catalytic production of O(2) by cytochrome b purified from porcine neutrophils in an artificial reconstitution system with an exogenous reductase. Purified cytochrome b had a midpoint reduction potential (E(m)) (^1)of -255 mV at pH 7.4 and showed low spin EPR signals at 10 K. In stopped-flow measurements, the reduced form of cytochrome b was reoxidized to the ferric form by O(2) according to a second-order reaction with a rate constant (k(2)) of 10^7M s at 10 °C. From these results, we have concluded that the heme in a six-coordinated low spin state catalyses one electron reduction of O(2).

All known heme-containing enzymes involved in reduction or activation of O(2), such as cytochrome c oxidase or cytochromes P-450, have a heme in which the five coordination sites of iron are occupied by intrinsic ligands, and the sixth coordination site is opened for binding O(2) or other extrinsic ligands. The heme of these enzymes forms an iron-O(2) complex during the reduction of O(2), or forms the complexes with respiratory inhibitors. On the other hand, the respiratory burst has been known to be insensitive to the respiratory inhibitors, and cytochrome bin situ does not form a complex with CO(15) . Judging from spectroscopic data, the heme of cytochrome b is in a low spin six-coordinate state in both the ferric and ferrous forms(12, 14, 15, 16, 17) . Moreover, we confirmed that the respiratory inhibitors gave no effect on the absorption spectra nor on the catalytic activity of purified cytochrome b in the reconstituted system(14) . These facts indicate strong coordination of both the axial ligands to the heme iron and suggest that cytochrome b reduces O(2) by a unique mechanism distinct from those of other heme-containing enzymes.

The purpose of this study is to examine whether an oxygenated intermediate is formed during the reduction of O(2) by ferrous cytochrome b or not and to clarify the mechanism of the reaction. The reduced cytochrome was mixed with an oxygenated solution under various conditions, and the spectral changes during the reaction were measured on the millisecond time scale by rapid-scanning spectroscopy. The temperature and pH dependence of the reaction was determined. Under anaerobic conditions, the ferrous cytochrome showed the ability to react with artificial oxidants. The properties of the reactions by cytochrome b are compared with those by other heme proteins. The results obtained here, together with our previous ones(13, 14, 15) , suggest that the reduction of O(2) by cytochrome b occurs via a peripheral process, i.e. electrons being transferred from the heme to O(2) at or near the heme edge without formation of the iron-O(2) complex.


EXPERIMENTAL PROCEDURES

Superoxide-producing cytochrome b was purified from porcine neutrophils according to the method described previously(14) , except that the gel filtration chromatography was performed with Superdex 200 pg (Pharmacia) packed into a 1.6 times 60-cm column. The final purification step with heparin affinity chromatography was omitted. The purified preparation was depleted of flavins (FAD/heme <0.01) as described previously(14) . The concentration of cytochrome b was determined using a reduced-minus-oxidized extinction coefficient of 21,600 M cm at 558 nm(18) .

Tetrazole myoglobin (Tet-Mb) was prepared and purified according to Shiro et al.(19, 20) . The time course of autoxidation of Tet-Mb was measured by following the absorbance change at 570 nm as described previously(19) .

Measurements of spectral changes of cytochrome b in a time range of milliseconds were carried out with a stopped-flow and rapid-scanning spectrophotometer, Unisoku type RSP-601. The purified cytochrome was diluted with a buffer solution containing 100 mM HEPES-NaOH, pH 7.4, and 0.1% sucrose monolaurate, unless otherwise noted in the text. The sample solution was depleted of O(2) by gently bubbling N(2) gas in one of the two reservoirs of the stopped-flow mixer. In the other reservoir, the buffer solution containing reagents mentioned in the text was bubbled with a gas mixture of N(2) and O(2), which was prepared with a Estec SGD-SC standard gas divider, for at least 15 min. In measurements of the reactions with artificial oxidants, both the solutions also contained 100 mM glucose, 10 units/ml glucose oxidase (Sigma), 2 units/ml catalase (Sigma), and 50 µg/ml superoxide dismutase (Sigma) and were bubbled with N(2) gas to completely remove O(2). The rapid-scanning recording of a spectrum took 1 ms/208 nm, and 512 spectra were measured with or without time intervals after mixing of the solutions. The dead time was estimated at about 6 ms based on the time course of reduction of dichlorophenolindophenol by ascorbate in separate measurements under the experimental conditions used. Concentration of O(2) in the reaction mixture was estimated by amounts of oxygenated myoglobin(Fe) formed after mixing deoxy myoglobin(Fe) with an oxygenated buffer in other separate measurements. Temperature was controlled by circulating water maintained at constant temperature with a Lauda thermostat type RM6 around the reserved solutions and the flow lines inside the mixer.

The time course data for the oxidation of ferrous cytochrome b by oxidants were analyzed according to the equation based on second-order reactions as described previously(14) . The change in concentration of cytochrome b from the ferrous form to the ferric form was estimated based on the reduced-minus-oxidized extinction coefficient at 426 nm of 110,000 M cm.


RESULTS

The ferric form of cytochrome b was rapidly reduced to the ferrous form with absorption maxima at 558, 528, and 425 nm after mixing with an excess amount of Na(2)S(2)O(4) under anaerobic conditions (Fig. 1). The spectral changes clearly showed isosbestic points at 417.4 and 438.2 nm. The time course data were fitted with a single exponential curve based on a pseudo-first-order reaction (not shown), and the apparent rate constant was estimated at 3.6 s under the experimental conditions (see the legend of Fig. 1).


Figure 1: Spectral changes during the reduction of cytochrome b with Na(2)S(2)O(4) under anaerobic conditions. Cytochrome b was mixed with a Na(2)S(2)O(4) solution at the final concentrations of 2.8 µM and 1 mg/ml, respectively, in a stopped flow apparatus, and the spectral changes were measured by a rapid-scanning spectrophotometer at 10 °C. The spectra recorded at 0, 60, 120, 180, 240, 300, 360, and 420 ms after the mixing are shown. Inset, the spectral changes in the Soret band are displayed as the difference spectra against that at 0 ms after the mixing.



The ferrous form of cytochrome b prepared by addition of a slight excess of Na(2)S(2)O(4) was mixed with an oxygenated solution prepared at a low concentration of O(2), and the spectral changes during the reaction of cytochrome b with O(2) were measured on the millisecond time scale (Fig. 2). The initial spectrum of the ferrous form was gradually replaced with that of the ferric form in the time domain. Clear isosbestic points were observed at 417.6 and 437.6 nm, which were identical with those in the spectral changes during the reduction with Na(2)S(2)O(4) under the anaerobic conditions within the wavelength resolution of the spectrophotometer (see Fig. 1).


Figure 2: Spectral changes during the oxidation of ferrous cytochrome b with O(2). Cytochrome b reduced with a small excess of Na(2)S(2)O(4) was mixed with an oxygenated solution at the final cytochrome and O(2) concentrations of 3.5 and 10 µM, respectively, and the spectral changes were measured at 2 °C. The spectra recorded at 0, 4, 8, 12, 16, 20, 40, and 80 ms after the mixing are shown. Inset, the spectral changes in the Soret band are displayed as the difference spectra against that at 80 ms after the mixing.



The effect of O(2) concentration on the time course for the reaction was examined as shown in Fig. 3, ad. The apparent rate increased with the O(2) concentration in the tested range. The addition of CO did not affect the reaction (Fig. 3, e). The second-order rate constants (k(2)) were almost constant under these conditions and were estimated at 8.29.5 times 10^6M s (see the legend to Fig. 3). Effects of the addition of CN, N(3), and pyridine were not observed (not shown).


Figure 3: Effects of O(2) concentration and addition of CO on the time course for the reaction of ferrous cytochrome b with O(2). The reactions were measured at 10 °C. Theoretical curves based on the second-order reaction were fitted to the data (dots) with the rate constants (k(2)) and the 0 ms concentrations of the ferrous cytochrome b as follows: a, 9.3 times 10^6M s and 1.13 µM; b, 9.5 times 10^6M s and 0.96 µM; c, 8.5 times 10^6M s and 0.69 µM; d, 8.2 times 10^6M s and 0.59 µM; e, 9.5 times 10^6M s and 1.05 µM, respectively. Addition of CO showed no inhibitory effect on the reaction (e). The absorbance at time 0 ms decreased with the concentration of O(2) by the oxidation during the dead time of the stopped-flow measurements (see ``Experimental Procedures'').



The effect of pH on the reaction of ferrous cytochrome b with O(2) was examined between pH 7 and 11. In this pH range, little change in absorption spectra for the ferric and ferrous forms was observed with the variation of pH. The rate constant of the reaction was almost constant over a pH range from 7 to 9, and decreased above pH 9 (Fig. 4). The pH dependence gives a single pK of 9.7.


Figure 4: Dependence of the rate constant on pH for the reaction of ferrous cytochrome b with O(2). The reactions were measured at 10 °C in buffer solutions which contained 0.1% sucrose monolaurate and 100 mM tris(hydroxymethyl)aminomethane-NaOH at pH between 7.8 and 9.0, 100 mMN-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid-NaOH at pH 9.4 and 9.9, or 100 mMN-cyclohexyl-3-aminopropanesulfonic acid-NaOH at pH 10.5 and 11. Initial concentrations of O(2) in the reaction mixtures were 10 µM at pH between 7.4 and 9.4, and 20 µM at pH between 9.9 and 11. Each data point is the average of 48 separate measurements, and error bars indicate the standard deviation from the average. The solid line was drawn assuming that k(2) decreased from 8.8 times 10^6 to 2.4 times 10^6M s with pK of 9.7.



The ability of ferrous cytochrome b to react with artificial oxidizing agents was examined under completely anaerobic conditions (Fig. 5). We found that cytochrome b was highly reactive with ferricyanide (E(m) = +420 mV). The spectral changes during the reaction (Fig. 5A) show clear isosbestic points, which are also observed in the reaction with O(2). k(2) for the reaction with ferricyanide was estimated at 6.4 ± 0.4 times 10^7M s (4 measurements) at 10 °C. In addition, p-benzoquinone (E(m) = +285 mV), a rather hydrophobic agent, was able to oxidize cytochrome b. The reaction, however, was much slower than that with ferricyanide (Fig. 5B), and k(2) was estimated at 3.5 ± 0.3 times 10^2M s (three measurements) at 10 °C.


Figure 5: Oxidation of ferrous cytochrome b by artificial oxidizing agents. A, spectral changes during the oxidation of ferrous cytochrome b with ferricyanide. Ferrous cytochrome b was mixed with an anaerobic solution containing ferricyanide at both final concentrations of 5 µM, and the spectral changes were measured at 5 °C. The spectra recorded at 0, 4, 8, 12, 16, 20, 40, and 100 ms after the mixing are displayed as the difference spectra against that at 100 ms after the mixing. B, time courses for the oxidation of cytochrome b by p-benzoquinone (trace b) and by ferricyanide (trace c) at 10 °C. Initial concentrations of ferrous cytochrome b, p-benzoquinone, and ferricyanide were 5 µM, 5 µM, and 5 mM, respectively. No absorbance changes were observed in the absence of oxidants (trace a), showing that the reaction mixture was completely depleted of O(2) under the experimental conditions. k(2) was estimated by fitting theoretical curves to these data (see text).



The temperature dependence of the reactions of ferrous cytochrome b with O(2) and ferricyanide was determined at pH 7.4 (Fig. 6). For comparison, the dependence of autoxidation of deoxy Tet-Mb was also determined under similar experimental conditions. The thermodynamic parameters of activation for these reactions were obtained on the basis of the transition state theory (21) and listed in Table 1with those for the reduction of ferric cytochrome c by O(2), which were calculated based on the data of Butler et al.(22) (see ``Discussion'').


Figure 6: Temperature dependence of the rate constant for the reactions of ferrous cytochrome b and Tet-Mb. The reactions of cytochrome b with ferricyanide (closed square) and O(2) (closed circle) and the reaction of Tet-Mb with O(2) (open circle) were measured at temperature between 2 and 40 °C at pH 7.4. Initial concentrations of O(2) were 10 and 350 µM for the reactions of cytochrome b and Tet-Mb, respectively, at 10 °C and were corrected according to the standard solubilization curve for the estimation of k(2). The reactions of cytochrome b with ferricyanide were measured under experimental conditions as described in the legend to Fig. 5except temperature.






DISCUSSION

From the k(2) value at 30 °C, the oxidation rate of ferrous cytochrome b by O(2) is estimated at about 8000 s under air-saturated conditions, which is hundreds of times faster than the rate of the steady state turnover, 120 s in the catalytic production of O(2)in situ or in our reconstituted system(13, 14) . The reduction of ferric cytochrome b under anaerobic conditions was considerably slower ((13) ). (^2)Furthermore, it is unlikely that a process following the reduction of O(2), such as product release, is rate-limiting because the lifetime of O(2), even in proteins, should not be so long as the heme reduction. Thus, the rate-limiting step in the catalytic reaction is the transfer of NADPH-derived electrons to the heme, and the single turnover reaction rather than the steady state one should be measured to investigate the properties of the reaction of cytochrome b with O(2) or to examine the effects of respiratory inhibitors.

The rate of the oxidation of ferrous cytochrome b linearly depends on the O(2) concentration, indicating that the reaction proceeds through a bimolecular process. During the reaction, the spectra of only the two (ferrous and ferric) forms of cytochrome b could be detected, and no intermediate species could be observed. This indicates that the reaction intermediate, if any, transiently forms and is rapidly decomposed to O(2) and the ferric heme. Otherwise, the absorption spectrum of the intermediate is indistinguishable from those of the ferrous and ferric forms under the wavelength resolution. Neither the spectra of the ferric and ferrous cytochrome nor the reaction with O(2) were affected by addition of CO, CN, and N(3). Artificial oxidizing agents, ferricyanide and p-benzoquinone, can react with ferrous cytochrome b apparently through an outer sphere mechanism. The rate of oxidation of ferrous cytochrome b by ferricyanide was even larger than by O(2). The thermodynamic analyses show that the set of the activation parameters for the reaction with ferricyanide is similar to that for the reaction with O(2), suggesting that the reaction with O(2) occurs by a mechanism similar to that with ferricyanide (Table 1, and also see below). The site for the reaction with ferricyanide appears to be located at a solvent accessible position on the protein and may be common with the site for the reaction with O(2). On the other hand, the site for the reaction with p-benzoquinone may be different from that for the reaction with O(2) and may be located at a position more distant from the heme. The higher redox potential of ferricyanide may contribute to the higher rate of the reaction. All of these results suggest that the coordination of both the internal axial ligands to the heme iron in cytochrome b is so strong that O(2) does not bind to the iron by replacing any internal ligand during the reaction and that the reaction with O(2) proceeds via an outer sphere mechanism.

Support for our idea mentioned above is provided by comparing the thermodynamic parameters in the oxidation of cytochrome b by O(2) and ferricyanide with the reactions of cytochrome c and Tet-Mb (Table 1). It was suggested that the reduction of ferric cytochrome c by O(2) takes place at or close to the solvent accessible heme edge on the basis of the thermodynamic and kinetic studies(22) . On the other hand, Tet-Mb, in which the distal histidine (His E7) of myoglobin is site-specifically modified with tetrazole anion (-CN(4)), can catalyze reduction of O(2) to O(2) in the presence of an enzymatic reduction system(19) . Tet-Mb exhibits a low oxidation reduction potential (E = -193 mV), which is comparable to that of cytochrome b, and has the six-coordinated heme iron in the ferric state by coordination of the tetrazole group as the internal sixth ligand(20) . In contrast with cytochrome b, the ferrous heme iron of Tet-Mb can bind CO and forms an iron-O(2) complex in the reduction of O(2). Therefore, the mechanisms for the reactions of cytochrome b and Tet-Mb with O(2) seem to mainly depend on the bond strength of the axial ligand to the sixth coordination site of the heme iron.

In any of these reactions listed in Table 1, the contributions of the entropic terms (TDeltaS) to the total energy barriers (DeltaG) are small and the enthalpic contributions (DeltaH) are predominant. However, DeltaH is much more predominant in the reaction by Tet-Mb than in those by cytochrome c and cytochrome b, whereas the TDeltaS values for the four reactions are similar to each other. The large DeltaH value for the reaction of Tet-Mb is similar to that for the autoxidation of native myoglobin by the formation of the iron-O(2) complex (85-150 kJ/mol)(23) . Since DeltaH is a measure of the energy barrier which must be overcome to break and make bonds in the formation of the transition state from the reactants, it seems reasonable that the lower enthalpic barriers in the reactions by cytochrome c and cytochrome b arise from the weaker interactions of O(2) with the ferric heme and of O(2) (or ferricyanide) with the ferrous heme, respectively, in these transition states.

The results obtained here, together with our previous data(13, 14, 15) , are best understood assuming an outer sphere mechanism for the reduction of O(2) by cytochrome b. The reaction can occur at or near the heme edge by a ``peripheral--process,'' which has been proposed for the autoxidation of coordinately saturated iron porphyrin complexes by Castro and co-workers(24, 25) . In this process, O(2) weakly interacts with the porphyrin plane of the ferrous heme followed by the electron transfer. The weak interaction between O(2) and the iron-porphyrin system is similar to those in peripheral complexes of metalloporphyrins with aromatic compounds(26) . In the heme of cytochrome b, the strong and symmetrical coordination of the axial ligands can enhance the interaction between iron d and porphyrin electrons to make the rapid O(2) reduction possible.

The weak interaction of O(2) with the heme of ferrous cytochrome b may be stabilized by hydrogen bonding with a basic amino acid residue near the heme pocket since the reaction slows down by increase of pH above 9 (Fig. 4) as follows (Fig. S1).


Scheme 1:


In this scheme, BH indicates the protonated basic residue. We have obtained no spectroscopic evidence for such a loosely associated complex of the heme with O(2). This seems to be inevitable since the formation of such a complex should be transient (half-time <1 ms) and/or the weak interaction can induce only slight changes in the absorption spectra that are not expected to be detectable under the experimental conditions.

It is also possible that a positive charge of an ionizable group near the heme rises the redox potential and gives higher reactivity with O(2) in the neutral pH range.

The major type of oxidases and oxygenases contains five-coordinated heme iron and catalyzes reduction of O(2) bound to its sixth coordination site to generate H(2)O or oxygenated products. Some of these enzymes also catalyze production of O(2) or H(2)O(2) in their uncoupled or abnormal reactions (27, 28, 29) . In the present study, we have suggested a novel mechanism for the reactions of the heme-containing oxidases. The mechanism for the O(2) reduction may be common in autoxidation of cytochromes with 6-coordinated hemes and also in bimolecular reactions of deoxy myoglobin and hemoglobin with O(2) under specific conditions (30, 31, 32) . It appears that phagocytic cytochrome b has evolved the coordination structure of the heme to be specifically adapted for the rapid production of O(2) even under low concentration of O(2).


FOOTNOTES

*
This work was supported in part by grants from the Ministry of Education, Science and Culture, Japan, by grants from Biodesign Research Program of RIKEN, and by Research Funds from the Science and Technology Agency, 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: Biophysical Chemistry Laboratory, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, Japan. Tel.: 048-462-1111 (Ext. 5465); Fax: 048-462-4660.

(^1)
The abbreviations used are: E(m), midpoint reduction potential; Tet-Mb, tetrazole myoglobin.

(^2)
Y. Isogai and Y. Orii, unpublished results.


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