©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytochrome bd Oxidase from Azotobacter vinelandii
PURIFICATION AND QUANTITATION OF LIGAND BINDING TO THE OXYGEN REDUCTION SITE (*)

Susanne Jünemann (§) , John M. Wrigglesworth (¶)

From the (1)Metals in Biology and Medicine Centre, Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cytochrome bd has been purified from Azotobacter vinelandii by a new simplified procedure. The heme and total iron content has been measured, as has the number of high affinity CO and NO binding sites. Spectral changes indicate high affinity binding of CO and NO to heme d only, with a stoichiometry of 1 molecule of gas per 2 molecules of heme b or per 3 atoms of iron. The results clearly define a stoichiometry of one heme d per complex. Low affinity binding of CO and NO to heme b595 also occurs at higher ligand concentrations. EPR heme-nitrosyl signals are seen with NO bound to both hemes b595 and d but with no indication of spin exchange coupling. Exposure of the air-oxidized complex to alkaline pH results in removal of molecular oxygen from heme d and a change in line shape of the high spin region of the EPR spectrum. Cyanide binds to both heme d and heme b595 in the air-oxidized complex, displacing molecular oxygen from heme d. The rate of cyanide binding to heme d as assessed by spectral changes at 650 nm does not correlate with the rate of binding to heme b595 as assessed by the loss of the high spin EPR signal. In addition, the cyanide binding rate in the presence of reductant is only 3 times that of the rate of binding to the air-oxidized enzyme, in contrast to the copper-containing oxidases where strong redox cooperativity makes these two rates differ by a factor of at least 10. The results do not support the idea of the presence of two strongly interacting hemes in a binuclear center.


INTRODUCTION

The cytochrome bd terminal oxidase complex of Azotobacter vinelandii is a quinol oxidase required for respiratory protection during aerotolerant nitrogen fixation(1) . It shows structural (2) and genetic (1, 3) similarities to the Escherichia coli enzyme, although the apparent oxygen affinity of the A. vinelandii complex is thought to be much lower(4) . The E. coli complex comprises two membrane-associated subunits of molecular mass 58 and 43 kDa(5) . Similar properties are reported for the A. vinelandii complex(6) . Cytochrome bd contains at least three redox-active centers: heme b558, heme b595, and one or two d-type hemes. EPR signals indicate two major high spin ferric heme signals attributed to b595 and heme d. A low spin signal has been assigned to heme b558(7) . Coulometric and CO binding studies on the E. coli enzyme (8) have suggested the presence of 2 mol of heme d. In support of this suggestion, there is evidence that the complex can bind simultaneously to molecular oxygen and peroxide(9) , and nitrate appears to bind to heme d with two distinct affinities(10) . However, the presence of two hemes d per complex has been questioned on the basis of EPR studies(7, 11) . CO binding studies on heme b595 have also given equivocal results, with optical measurements and potentiometric titrations appearing to differ(12, 13) . Unfortunately, the use of optical spectroscopy to assign the absorbance bands in the Soret region is difficult, mainly because of band overlap and weak absorption, but in the -band region three heme types have been distinguished(8) .

The cytochrome bd complex is of particular interest not only because of its role in respiratory protection in A. vinelandii, where its enzyme activity is one of the highest of all the terminal oxidases, but also because the mechanism of oxygen reduction can be contrasted with that of the copper-containing oxidases. These latter oxidases also reduce oxygen to water in a binuclear heme/copper center with strong cooperativity between the two redox metals. Several well defined intermediates have been detected in this center(14) . The copper-containing oxidases have the additional capability of proton translocation(15) , which is thought to involve coordination changes in or around the copper/heme binuclear center(16, 17) . In comparison, little is known about the reaction mechanism of the cytochrome bd oxidase, which shares no primary sequence homology with the copper-containing oxidases although mechanistic similarities exist. Oxygenated and peroxy states have been detected (18, 19) as well as an oxoferryl heme d(20) . The reconstituted system is capable of forming a transmembrane electrochemical gradient, although without translocation of protons(21) . At the oxygen reduction site, interaction between the high spin heme groups, b595 and d, has been suggested by EPR studies(7) . CO titrations (22) and Fourier transform infrared spectroscopy (23) have led to the proposal of a heme-heme binuclear center at the oxygen binding site. In the present work, we have purified and characterized the complex from A. vinelandii in order to quantitate the binding of various ligands at the oxygen binding site. The results clearly show the presence of one heme d per cytochrome bd complex. In addition, the results of CO, NO, and CN binding argue against the presence of a strongly interacting binuclear center in the enzyme.


EXPERIMENTAL PROCEDURES

Purification of the Cytochrome bd Complex

The cytochrome bd-overexpressing A. vinelandii strain MK8 was grown essentially as described by Kelly et al.(1) . All steps were carried out at 4 °C. Membranes from approximately 50 g, wet weight, of cells were prepared (1) and suspended to a protein concentration of 10 mg/ml in 25 mM Tris/HCl, pH 8.0, containing 10 mM EDTA and 0.5 M KCl. The membranes were pelleted at 100,000 g for 1 h and resuspended in 0.1 M potassium phosphate buffer, pH 7.2 (10 mg of protein/ml). Sodium cholate was added from a 10% (w/v) stock solution to give a final concentration of 2% (w/v), and the mixture was left stirring overnight. After centrifugation at 45,000 g for 20 min, the cytochrome bd-containing pellet was washed twice in phosphate buffer alone to remove the remaining cholate. The pellet was suspended in 0.1 M phosphate buffer, pH 7.2, (15 mg of protein/ml), and lauryl maltoside was added to a final concentration of 1.5% (w/v). The suspension was stirred for 1 h before being centrifuged at 45,000 g for 15 min. Ammonium sulfate was added to the supernatant to give 55% saturation. The mixture was left for 30 min and centrifuged at 45,000 g for 15 min. The red pellet was discarded. Raising the ammonium sulfate concentration to 80% and adjusting the pH to 7.2 with dilute ammonium hydroxide precipitates the oxidase complex. The floating pellet was dissolved in 0.1 M phosphate buffer, pH 7.2 (10-15 mg of protein/ml) and stored under liquid nitrogen.

Chemicals and Assays

The protein concentration of vesicle suspensions and membrane protein preparations was measured by a modified Lowry method(24) . Ubiquinone-1 was a kind gift from Dr. R. Cammack (King's College London). Before use, it was chromatographed on Merck silica gel 60 TLC plates, using chloroform:hexane:diethylether 10:10:1 as solvent. The ubiquinone-1 was then dissolved in ethanol. The assay for oxidase activity was based on that described by Kita et al.(25) using a Clark-type oxygen electrode at 25 °C containing 1 ml of 0.1 M potassium phosphate, pH 7.9, plus ubiquinone-1 and DTT()at the concentrations indicated. The reaction was started by adding 5-10 µg of cytochrome bd that had been pre-incubated on ice for 5 min in 10 µl of 0.1 M phosphate buffer, pH 7.9, containing 1 mM phospholipid (L--phosphatidylcholine from Soybean, Type IV-S, Sigma). Optical spectra were recorded on a Varian Cary 210 spectrophotometer at room temperature, using a spectral bandwidth of 1 nm and a scan rate of 2 nm s. The spectra were stored in digitalized form for treatment. SDS-polyacrylamide gel electrophoresis was performed in a minigel unit (10 7.5 cm) essentially as described by Laemmli (26) with the exception that the sample buffer contained 6% (w/v) SDS and 8 M urea in order to prevent aggregation of the oxidase complex. The samples were loaded onto the gel without prior incubation. Gels were stained with Coomassie Brilliant Blue (2% in 45% (v/v) methanol, 10% (v/v) acetic acid) and destained in the same solvent without dye. Pyridine hemochrome spectra were determined by mixing 0.75 ml of diluted cytochrome preparation with 175 µl of pyridine and 75 µl of 1 M NaOH. The dithionite-reduced minus ferricyanide oxidized difference spectrum was immediately recorded. The b-type hemes were quantified using an extinction coefficient of 24 mM cm (556 nm minus 540 nm)(27) . Continuous wave EPR measurements were recorded on a Bruker ESP300 spectrometer fitted with a TE103 rectangular cavity and a Hewlett-Packard microwave frequency counter 5350B and an Oxford instruments liquid helium flow cryostat ESR900. Spectra were base line-corrected by subtraction of a cavity spectrum of water/buffer under identical conditions. The iron and copper content of the samples was determined by atomic absorption spectroscopy using a Varian AA-1475 spectrometer.

Carbon Monoxide and Nitric Oxide Binding Titrations

Nitric oxide was generated by reaction of 2 M sulfuric acid with sodium nitrite under nitrogen atmosphere and washed by passage through 1 M sodium hydroxide and water. Saturated solutions of carbon monoxide or nitric oxide were prepared by bubbling the respective gas through ice-cold water for 10 min. The concentration of the gas in solution was determined by titration of a myoglobin standard. Aliquots of the stock solution were added anaerobically to the reduced cytochrome preparation at room temperature. After equilibration (2-3 min), the spectrum was recorded over the range 400-700 nm. The end point of a titration was determined by bubbling the gas through the suspension at room temperature to obtain a saturated sample.


RESULTS

Purification of the Cytochrome bd Complex

Cytochrome bd has been purified previously from E. coli(5, 25) , Klebsiella pneumoniae(13) , Photobacterium phosphoreum(28) , and A. vinelandii(6) . The experimental conditions for the first three organisms cannot be directly applied to A. vinelandii, mainly because of the instability of the complex in the detergents used. We have developed a simple and fast procedure for purification of the complex from the overexpressing mutant strain MK8 of A. vinelandii based on ammonium sulfate precipitation. This was briefly described by Jünemann and Wrigglesworth(29) , and a full characterization of the preparation is given here. summarizes the results of a representative preparation of the cytochrome bd complex. The initial wash in KCl removes flavin and b/c-type cytochromes as judged from the optical spectra (results not shown). The crucial step appears to be the extraction in lauryl maltoside, where the oxidase is solubilized selectively. Any extra contaminating b-type cytochrome is precipitated by the subsequent ammonium sulfate treatment which was also shown, by EPR, to remove iron-sulfur complexes. An SDS gel of the final preparation (Fig. 1) shows the presence of two main bands indicative of polypeptides with molecular masses of 50 and 31 kDa. These values can be compared with values of 50.1 and 35 kDa as calculated from the nucleic acid sequences of the two subunits of cytochrome bd from A. vinelandii. (It should be noted that the values of 59.7 and 42 kDa quoted in the literature for this calculation (30) are higher because no allowance for water removal on peptide bond formation was made.) Components with other molecular weights comprised less than 12% on the basis of densitometry scans of Coomassie-stained gels. A pyridine hemochrome spectrum of the purified complex (not shown) revealed only b-type (556 nm) and d-type (608 nm) hemes to be present. Quantitation of the hemes by this method depends on the extinction coefficient for the pyridine hemochromes. No reliable value has been found for d-type heme but an early value of 20.7 mM cm for b-type heme (41) has been remeasured as 24.0 mM cm(27) . Using this latter figure, the present preparation contains 10.3 nmol of heme b/mg of protein (). The iron content determined by atomic absorption spectroscopy was found to be 17.6 nmol/mg of protein (), which is 1.7 times the amount of the total b-type heme content determined by the pyridine hemochrome method. As expected, no copper was detected in the final preparation. The final preparation oxidized ubiquinol-1 (ubiquinone-1 plus DTT) with specific activity similar to that of the preparation described by Kolonay et al.(6) . The activity with alternative substrates was lower, being 230 nmol of O consumed/s/mg of protein using duroquinol (250 µM plus 1 mM DTT) and 127 nmol O consumed/s/mg of protein with N,N,N`,N`-tetramethyl-p-phenylenediamine (1 mM plus 5 mM ascorbate).


Figure 1: SDS-polyacrylamide gel of purified cytochrome bd complex from A. vinelandii. Gel dimensions: 7.5 10 0.075 cm, with loadings of 2 and 4 µg of protein.



Spectroscopic Properties

The absolute spectra of air-oxidized and dithionite-reduced complex are shown in Fig. 2together with the difference spectrum. The main features are similar to those of the cytochrome bd complexes purified from P. phosphoreum(28) , E. coli(9) , and K. pneumoniae(13) . The samples as prepared contained a high proportion of the stable oxygenated species with a peak in the air-oxidized spectrum at 646 nm (trough in the difference spectrum at 650 nm). An indicative measure of the purity of the complex is the ratio of peak intensities in the reduced minus air-oxidized spectra at 560-580 nm and 630-650 nm, which approaches 1 for the purer preparations. The slight shoulder around 550 nm is not due to c-type cytochrome but has been ascribed to the -band of heme b595 in E. coli(21, 31) . The EPR spectrum of the air-oxidized purified complex is shown in Fig. 3(uppertrace). Simulation of the spectrum in the high spin region suggests the presence of both axial and rhombic components. A prominent high spin axial component at g = 6 comprises over two-thirds of the signal and, in the E. coli complex, has been assigned to heme d(7, 11) . The remaining high spin rhombic features at g = 5.5 and g = 6.3 have been assigned to heme b595. Features associated with ferric low spin heme can be seen at g = 3.4 and around g = 2.55. The g = 3.4 signal has been shown in E. coli to titrate with an E of 226 mV and has been assigned to heme b558(7) . Signals around g = 2.5 in the E. coli enzyme have been resolved into a combination of three different components including a rhombic form with g and g features at 2.61 and 2.48(11) . The intensity of the signals was found to vary from preparation to preparation, but in redox titrations they all titrated with the same redox midpoint potential and have been ascribed to low spin ferric heme d(11) . We ascribe the signal around g = 2 in the present preparation to a small amount of free radical with a signal close to the g component of the high spin axial signal. A small amount of nonspecifically bound iron can be seen at g = 4.3, consistent with the iron content measured by atomic absorption being slightly higher than 1.5 times the total b-type heme, assuming a b/d heme ratio of 2:1.


Figure 2: Absorption spectra of purified cytochrome bd complex from A. vinelandii. Samples contained 3.1 µM oxidase complex in 0.1 mM potassium phosphate, pH 7.2. Air-oxidized (solidline), dithionite-reduced (dashedline), and difference spectra (dottedline) are shown.




Figure 3: EPR spectra of the purified cytochrome bd complex from A. vinelandii. The air-oxidized samples (26 µM) were suspended in potassium HEPES, pH 7 (uppertrace) and in Tris-HCl, pH 9 (1-min exposure) (lowertrace). Conditions of measurement were as follows: temperature, 5 K; microwave frequency, 9.35 GHz; microwave power, 20 mW; modulation amplitude, 10 G; modulation frequency, 100 kHz; gain, 2 10; time constant, 163 ms. The spectra are base line-corrected by subtraction of the spectrum of buffer measured under the same conditions. Simulated spectra (program by Neese (45)) are shown as dottedlines. The parameters used in the simulations were as follows: rhombic component, g, g, and g at 6.20, 5.47, and 2.00, respectively, with linewidths 16.0 G, 19.5 G, and 100 G, and axial component, g, g, and g at 5.915, 5.88, and 2.00, respectively, with linewidths 9.5 G, 40 G, and 100 G. See ``Results'' for the relative contributions of each component to the final spectra.



Hydroxide Effects on the Air-oxidized Cytochrome bd Oxidase

Fig. 3(lowertrace) shows the effect of short time exposure at alkaline pH on the EPR spectrum of the air-oxidized bd complex. The main change is a decrease in the high spin axial component and an increase in the rhombic signal. Changes in the optical spectrum at 646 nm (not shown) indicated that deoxygenation of heme d occurred at alkaline pH. Simulation of the EPR spectra (Fig. 3, dottedlines), assuming no pH-dependent change in the zero field-splitting parameters for the axial and rhombic components, indicates that the total intensity of the high spin components drops by approximately one-third at alkaline pH. However, most of this drop is due to a loss of the axial component. The relative amount of the rhombic species approximately doubled (from 28 to 74%), corresponding to an absolute increase by a factor of 1.8. It would appear that a pH-dependent removal of molecular oxygen from oxygenated heme d is associated with a change in the line shape of the high spin signal in the EPR spectrum. No significant changes were detected in the low spin region. In particular, no signals indicative of the formation of any low spin ferric complex with hydroxide (S = ) could be identified. The EPR changes were fully reversible on resuspending the sample back in pH 7 buffer after ultrafiltration through an Amicon YM10 membrane.

CO Binding to Reduced Cytochrome bd

The CO reduced minus reduced difference spectra of the purified complex at low (3 µM) and saturating (>0.8 mM) CO concentrations are shown in Fig. 4. Binding of CO induces changes in the -band spectrum of reduced heme d. Titration of these changes (644 nm minus 623 nm) shows saturation at low (stoichiometric) amounts of CO (Fig. 5A), giving an average of 5.1 ± 0.4 (n = 4) nmol of CO bound/mg of protein, equivalent to 1 CO bound/complex (). The difference spectrum in the Soret region for these titration experiments is more complex (Fig. 4). A clear trough can be seen at 443 nm, but also present is a double trough between 415 and 426 nm. At higher CO concentrations, no further changes occur in the heme d spectrum at 644 nm minus 623 nm, but a deepening of the trough at 426 nm occurs together with smaller changes at 560 nm and 590-600 nm. We attribute these changes to CO binding with low affinity to heme b595.


Figure 4: CO reduced minus reduced difference spectrum at low (3 µM) (A) and high (approximately 1 mM) (B) CO concentrations. The samples were reduced by dithionite. Other conditions of measurement were as in Fig. 2.




Figure 5: CO and NO binding titration to purified preparations of the reduced cytochrome bd complex. Experimental details were as described under ``Experimental Procedures''. A, CO binding as monitored by following using 0.44 mg ml complex the spectral changes in the -band region of oxygenated heme d (A). B, NO binding using A with 0.86 mg ml complex.



NO Interaction with the Reduced Cytochrome bd Complex

The optical difference spectrum of NO interaction with dithionite-reduced cytochrome bd is shown in Fig. 6. A titration of the redshift of the heme d -band on NO binding (653 nm minus 627 nm in the difference spectrum) results in 5.5 ± 0.4 nmol of NO bound/mg of protein (Fig. 5B). This high affinity binding is associated with changes in the Soret region of the optical spectrum at 443 nm and around 425 nm. On increasing the NO concentration to higher than stoichiometric amounts, features develop at 437, 560, and 595-600 nm, which we attribute to low affinity binding to heme b595. Binding of NO to reduced cytochrome bd also induces a heme-nitrosyl EPR spectrum (Fig. 7, tracea) with some similarities to that reported for the NO complex of the -subunit of hemoglobin(42) . The nature of this spectrum changes as the NO concentration is increased from stoichiometric amounts to higher levels (Fig. 7, traceb) consistent with NO binding to a separate heme site. Subtraction of the two spectra from each other (Fig. 7, uppertrace) shows the low affinity species (appearing at high NO concentrations) to have strong rhombic symmetry similar to that seen in NO derivatives of several ferrous hemeproteins (42) including the -subunit of hemoglobin(43) . A well resolved hyperfine structure was not obvious under the conditions used for either the low NO affinity or high NO affinity spectra. Samples for EPR were taken alongside the optical titrations, at low and high ligand concentrations. When quantified against a copper/EDTA standard, the spin concentration at stoichiometric amounts of NO was 5.2 nmol/mg of protein, increasing to 10 nmol/mg of protein at high NO concentrations. We conclude that there are two NO binding sites of equal concentration but different affinities. The spectral evidence indicates that these are heme d and heme b595, respectively. No obvious evidence for strong spin coupling between these hemes can be seen. This contrasts with the cytochrome c oxidases, where the nitrosyl signal from NO-ligated heme a disappears when NO binds to the close by Cu(32) .


Figure 6: The optical spectrum of NO interaction with dithionite-reduced cytochrome bd complex (0.86 mg ml) in 0.1 M potassium HEPES, pH 7. A, 7.5 µM NO concentration; B, 1-min bubbled NO through the sample.




Figure 7: EPR spectra of NO-treated dithionite-reduced cytochrome bd complex. NO and cytochrome bd concentrations were as in Fig. 6. EPR conditions were as in Fig. 3 except the temperature was 77 K, and the microwave power was 6.3 mW.



The Interaction of Cytochrome bd with Cyanide

Previous studies using membrane particles from E. coli and A. vinelandii have shown that the rate of cyanide binding to cytochrome bd oxidase is more rapid at higher rates of respiratory activity(33, 34) . In the present case, binding of cyanide to the purified complex under turnover conditions, with ubiquinol as substrate, occurs approximately 3 times faster (second order rate constant of 2.6 M sec) than the rate of binding of cyanide to the air-oxidized complex, as assessed by spectral changes at 650 nm (Fig. 8). From Fig. 9it can be seen that the final species formed is a ferric heme d-cyanide complex. A loss of oxygenated heme d occurs in the air-oxidized enzyme (Fig. 9A), and cyanide prevents the formation of ferrous heme d when added in the presence of reductant (Fig. 9B). These results suggest that the difference between the rates of cyanide binding to the complex in the absence and presence of respiratory substrates is due to the faster removal of molecular oxygen from heme d under turnover conditions and/or a higher steady-state level of the actual CN binding form of heme d. Krasnoselskaya et al.(35) have suggested concerted binding of cyanide to ferric hemes d and b595, possibly as a bridging ligand. If this does occur in the air-oxidized enzyme, the present results show that cyanide binding to ferric heme b595 is not strong enough to prevent its reduction, in contrast to the cyanide-bound heme d species, which remains ferric in the presence of reductant. In the EPR spectrum (Fig. 10), there is a drop in both the axial and rhombic components of the g = 6 signal when cyanide binds to the air-oxidized cytochrome bd complex. The intensity falls by approximately 30% after incubation with 10 mM cyanide for 1 h and continues to fall with longer incubation. It is clear that the full effect of cyanide on the high spin signal is much slower than the binding of cyanide to heme d as assessed by the optical changes at 650 nm. On the other hand, 1 h of incubation with cyanide completely removes the g = 2.55 signal, and a new signal appears at g = 2.97 (Fig. 10). Kauffman et al.(44) report a value of g = 2.99 for the position of the heme d ferric cyanide signal in membrane preparations from A. vinelandii incubated with 5 mM cyanide. A smaller feature (at g = 3.23) was also noted by these workers. In the present case, small but significant perturbations can also be seen in the spectrum around the g = 2.97 signal in the cyanide-treated sample. When heme b595 is reduced these are lost together with the g = 3.4 signal from b558. The g = 2.97 signal remains, confirming the findings of the optical spectra (Fig. 9) that the low spin ferric cyanide complex of heme d is not reduced by ubiquinol.


Figure 8: Rate of cyanide binding to oxygenated heme d in the absence () and presence () of respiratory substrates. Conditions of measurement were as follows: cytochrome bd (2.1 µM) in potassium phosphate (0.1 M), pH 7.2, ubiquinone-1 (250 µM), and DTT (3 mM).




Figure 9: Optical difference spectra of cyanide treated air-oxidized cytochrome bd complex. Concentrations were as in Fig. 8. Samples were incubated with cyanide for 30 min. A, cyanide-treated minus air-oxidized complex; B, cyanide-treated complex in the presence of ubiquinone-1 (250 µM) and DTT (3 mM) minus air oxidized complex; C, control sample, reduced (ubiquinone-1 and DTT for 30 min) minus air-oxidized complex.




Figure 10: EPR spectra of cyanide binding to purified cytochrome bd oxidase. Conditions of measurement were as in Fig. 3. Other conditions were as in Fig. 9.




DISCUSSION

Purification of the Cytochrome bd Complex

The first successful purification of cytochrome bd from A. vinelandii was reported by Kolonay et al.(6) , who used DEAE-Sepharose chromatography in the presence of dodecyl maltoside. The present procedure provides a lower cost alternative based on ammonium sulfate precipitation, which gives a final preparation of comparable yield, purity, and activity. The final concentration of b-type heme shown in is 10.3 nmol/mg of protein. If a molecular mass of 85,100 Da is used for the molecular mass of the complex from A. vinelandii, as derived from the nucleic acid sequences for subunits I and II, then a theoretical value can be calculated for the concentration of total heme b/mg of purified complex. This turns out to be approximately twice the measured value of the present preparation and also for the final preparation of Kolonay et al.(6) . Both methods used the overproducing strain of A. vinelandii MK8. Contaminating protein of molecular weight different from the two main bands on the electrophoretic gel was estimated at no more than 12%. We suggest that a significant amount of (totally heme-free) apoprotein is also present in these membranes and copurifies with the active complex. Although this would lower the apparent value of heme per mg of complex, the relative stoichiometry of the different hemes per complex would be unaffected.

The thorough characterization of the purified complex provides a consistent heme stoichiometry from several independent methods (µM heme b from pyridine hemochrome spectra, µM iron from atomic absorption, µM high affinity binding of CO and NO) and can now be used to provide a reliable extinction coefficient in the -region of reduced heme d. A value of 12.0 ± 1.0 mM cm for the wavelength pair 628 nm minus 605 nm was calculated for the reduced minus air-oxidized complex. It should be noted that this wavelength pair is unaffected by the amount of oxygenated species present. The corresponding value reported for the E. coli enzyme is 7.4 mM cm(5) .

Stoichiometry of the Redox Centers

The apparent simplicity of the cytochrome bd complex belies many experimental anomalies. The first concerns the number of redox centers. It is generally assumed that the complex incorporates two classes of redox-active heme, b and d. Previous experiments have clearly identified two species of b-type heme, heme b558 and heme b595(5, 7) . However, 2 mol of heme d/complex have been suggested on the basis of CO titration experiments(8) , and support for this stoichiometry is provided by the presence of two binding affinities of nitrite to heme d(10) . The present data from metal and heme analysis, as well as ligand binding titration results are summarized in . The ratio of total iron to b-type heme is 1.7. This result indicates the stoichiometry of two b-type hemes plus one remaining heme, namely heme d. The small excess of iron, in addition to an expected value of 1.5 Fe/b-heme, may be due to some nonspecific iron as indicated by the EPR spectrum at g = 4.3. This stoichiometry is further supported by the results of CO and NO titrations, which do not depend on quantitation of b-type heme. Both CO and NO bind with high affinity to reduced heme d at a stoichiometry of 1 mol of ligand/mol of complex. With this binding, the optical absorbance of reduced heme d is fully removed. Finally, the spin concentration of the high affinity heme-nitrosyl signal gives a value of one per complex. We therefore conclude that cytochrome bd contains only one heme d per complex.

Ligand Binding to Hemes b595 and d

Although CO and NO bind to reduced heme d with high affinity, some low affinity binding to reduced heme b595 also takes place. It is possible to use these different affinities to identify spectral changes in the Soret region due to the two hemes. Changes in the Soret region with high affinity binding of CO and NO indicate that the weak band at 443 nm must represent changes in heme d absorption alone. The assignment of the Soret band of reduced heme d to 443 nm would be in agreement with the findings of Poole et al.(18) , who noted a small change at 445 nm following low temperature photolysis of bound CO in E. coli. Flash experiments using a helium/neon laser at 632.8 nm (36) have also indicated that a single absorbance band in the Soret spectrum at 443 nm can be assigned to heme d. High affinity CO binding also induces spectral changes at 415 nm and around 540 nm (Fig. 4). Similar changes have been noted in low temperature CO-photodissociation spectra in the presence of oxygen by d'Mello et al.(4) . These changes are not seen with NO binding (Fig. 6). At present we have no obvious explanation for these effects. The parallel changes around 595 nm and in the Soret region, following low affinity binding of CO or NO, indicate binding to heme b595 although the position of the trough in the Soret region is not the same for the two ligands.

Cyanide has been shown to induce a large red shift in the Soret peak of air-oxidized complex of E. coli, and some of this change (up to one third) is reported to take place before any significant development of the trough at 648 nm(35) . On the basis of MCD evidence, Krasnoselskaya et al.(35) conclude that up to two-thirds of the air-oxidized complex purifies in the oxygenated state, which prevents fast cyanide binding to either heme b595 or heme d. The present results show that the addition of substrate to the enzyme increases the rate of cyanide binding to heme d, consistent with a faster removal of molecular oxygen from this heme under turnover conditions. What is interesting to note is that cyanide binding in the presence of substrate is only approximately 3 times faster than under non-turnover conditions. This contrasts with mitochondrial cytochrome c oxidase, where there is strong redox interaction between the redox centers and the rates of binding of cyanide to the oxidized and partially reduced enzyme differ by a factor of 10 (37).

Full displacement of molecular oxygen occurs with both cyanide binding and exposure to alkaline pH in the air-oxidized complex. However, after incubation with cyanide for the same time period as used for measuring the optical changes, the loss of spin intensity in the high spin region of the EPR spectrum is less than one-third. We therefore suggest that in the air-oxidized complex, heme b595 is predominantly responsible for the high spin signals and that most heme d in air-oxidized enzyme is in the oxygenated form. Resonance Raman studies have shown that the oxygenated form of cytochrome d is very similar to oxyhemoglobin(38) . Indeed, removal of oxygen from the air-oxidized complex by argon treatment produces a partially reduced state(9) . This would then indicate an EPR silent Fe(II) ground state for the oxygenated heme d. Previous quantitations vary in the amount of the oxygenated form present(9, 35, 39) , but most estimates give values between 70-100%. The present results with hydroxide and cyanide treatment indicate that some of the remaining heme d is ferric and contributes between 20-30% to the spin intensity at g = 6. (This should not be confused with the EPR signals from oxidized preparations under anaerobic conditions, where almost 100% of the heme d has been shown to be ferric high spin and axial(7) .) With cyanide treatment, a new low spin signal appears with the formation of the cyanide complex of ferric heme d. It is interesting to note that the low spin signal around 2.55, assigned to heme d(11) , disappears on cyanide ligation, indicating that a small amount of low spin ferric heme d is present in the air-oxidized preparation. A more complicated picture arises for hydroxide treatment. The formation of a low spin ferric heme-hydroxide complex might be expected to give rise to an easily detectable EPR signal (S = ). None could be seen under the conditions of the present experiments. Direct ligation of hydroxide to ferric heme d does not seem to occur despite total loss of oxygen from ferrous heme d. The low spin signal at g = 2.55 from ferric heme d is still present at alkaline pH. Rothery and Ingledew (7) have noted that alkaline pH lowers the total intensity in the high spin region of the spectrum by approximately 20% and drastically increases the relative size of the rhombic component. Unfortunately the low spin region of the EPR spectrum was not presented, but the authors suggested that there is a pH-dependent equilibrium between the rhombic and axial components of the aerobic spectrum. This equilibrium would be affected as changes in pH titrate an ionizable group associated with heme b595. The present results indicate that this must occur without direct ligation of hydroxide to heme.

The Nature of the Oxygen Binding Site

Previous work on CO binding (29) and CO photodissociation (23) has provided some evidence for the presence of a heme-heme binuclear center in the cytochrome bd complex. Some interaction between heme b595 and heme d is also indicated from the fact that binding of molecular oxygen to anoxically oxidized heme d perturbs the EPR line shape of heme b595(7) . Similar perturbations are seen in the present work when oxygen is removed from heme d by exposure to alkaline pH, but as discussed above this may simply be due to the effects of changes in an ionizable group close to heme b595. The results with NO and cyanide binding show that any cooperative interaction between the redox centers in cytochrome bd must be weak. There is no strong anti-ferromagnetic coupling when NO is bound to both heme b595 and heme d. The apparently simple EPR characteristics of NO binding are good evidence for two binding sites that are some distance from each other and hence not part of a binuclear center. Spectral evidence clearly identifies heme d and heme b595 as the two sites. The close distance exchange coupling found in the copper-containing oxidases is not present in cytochrome bd and is probably the reason why the oxygenated heme d species is so stable. In cytochrome aa, this species is very short lived at room temperature(40) . Similarly, cyanide binding to heme d is only slightly influenced by the presence of substrate. This contrasts to the cyanide affinity of heme a in cytochrome c oxidase, which is greater by over a factor of 10 in the partially reduced enzyme compared with the fully oxidized or fully reduced states(37) . Overall, the results argue against the presence of a binuclear center where the two hemes act in a strong cooperative manner to facilitate oxygen reduction.

  
Table: Purification of cytochrome bd from A. vinelandii

See ``Experimental Procedures'' for details of purification stages.


  
Table: Analysis of purified cytochrome bd complex from A. vinelandii

Analysis and conditions of measurement were as described under ``Experimental Procedures.'' The number of determinations is given in parentheses.



FOOTNOTES

*
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.

§
Supported by the Biotechnology and Biological Sciences Research Council.

To whom correspondence should be addressed. Fax: 44 171 333 4500; E-mail: UDBC042@HAZEL.CC.KCL.UK.

The abbreviation used is: DTT, dithiothreitol.


ACKNOWLEDGEMENTS

We thank Dr. S. Hill (Nitrogen Fixation Laboratory, University of Sussex) for growth of A. vinelandii cultures and Dr. Steve Rigby for helpful discussions.


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