Azide binding to cytochrome c oxidase

369

Bioelecirochemistry and Bioenergetics, 17 (1987) 369-381 A section of J. Electroanal. Chem., and constituting Vol. 231 (1987) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

AZIDE BINDING TO CYTOCHROME

974 -

EFFECTS ON THE OXIDATION-REDUCTION PROPERTIES OF CYTOCHROMES Q AND

J. GORDON

LINDSAY

Department

of Biochemistry,

CHARLES

S. OWEN

Department

of Biochemistry,

DAVID

AND SPECTRAL Q3

University of Glasgow, Glasgow, Scotland (Great Britain)

Jefferson

Medical College, Philadelphia,

PA 19107 (U.S.A.)

F. WILSON

Department (Revised

c OXIDASE

of Biochemktry

manuscript

and Biophysics,

received

October

University of Pennsylvania,

Philadephia,

PA 19104 (U.S.A.)

23rd 1986)

SUMMARY The azide dependence of the properties of the hemes of cytochrome c oxidase has been evaluated in preparations of submitochondrial particles from pigeon breast muscle. Anaerobic suspensions were subjected to potentiometric titrations in the presence and absence of azide. When azide was present, the contribution of the high potential heme increased from 50% to 87% of the total absorption change at 603-624 nm with little change ( < 1 nm) in the absorption maximum. The low potential heme decreased in total alpha band absorption and its absorption maximum shifted from 606 nm to 610 nm. The two hemes made approximately equal contributions to the Soret absorption peak and this changed only slightly when azide was added. In the presence of a constant azide concentration of 20 mM, the high potential heme had an essentially unchanged half-reduction potential at pH 7.0 (U,,,,,O) but became less pH dependent at more alkaline pH values (30-50 mV/pH unit “errus 60 mV/pH unit), while the low potential component became pH independent. The possible identification of the hemes observed in the presence of azide with cytochromes a and a3 is discussed.

INTRODUCTION

The interaction of ligands such as HCN, CO, or HN, with the cytochrome c oxidase region of the mitochondrial respiratory chain has been the subject of intensive investigation. Analyses of the kinetics of binding, resolution of the inhibitory site and interpretation of the induced spectral alterations on cytochromes a and a3 are essential to understanding the mechanism of this important enzyme. 0302-4598/87/$03.50

0 1987 Elsevier Sequoia

S.A

Cytochrome c oxidase contains four redox components, cytochrome a, low potential (visible) copper, high potential (invisible) copper and cytochrome a3; measured properties of these components have indicated that the two cytochromes are strongly interactive [l-8]. This interaction is evident from measurements of light and e.p.r. absorption spectra and redox properties of each cytochrome, which are reported to be dependent upon the redox state of the other cytochrome [l-8]. Moreover, evidence has been presented that the high potential copper associated with cytochrome a, is an integral part of the oxygen reaction site and is involved in binding of oxygen [9,10], carbon monoxide [9-131, and nitric oxide [14]. In the present paper we report quantitative measurements of azide binding to cytochrome c oxidase as expressed by its effects on the redox and spectral properties of cytochromes a and ug. These data are then utilised to evaluate the ligand binding properties of cytochromes a and a3 as well as the interactions between these two cytochromes. MATERIALS

AND METHODS

Submitochondrial particles from pigeon breast mitochondria were prepared as described previously [15]. Simultaneous oxidation-reduction verSu.s absorbance titrations were performed essentially as described by Dutton and co-workers [15,16], employing dilute solutions of dithionite for addition of reducing equivalents. Oxidative titrations were performed by adding aliquots of 100 PLM potassium ferricyanide. Spectra of the difference in absorption from samples at different oxidation-reduction potentials were recorded using a Johnson Research Foundation dual wavelength spectrophotometer fitted with a scanning attachment, employing a spectral bandwidth at half height of less than 1 nm. A previously recorded absorption spectrum of a sample under one set of conditions could automatically be subtracted from a scan of the sample under a different set of conditions by means of a digital memory unit with the capacity to store two spectra. All reagents employed were of the highest grade available commercially. RESULTS

The pH dependencies of the half-reduction potentials of cytochromes a and a3 in pigeon breast submitochondrial particles (SMP) in the absence of added ligands are shown in Fig. 1 and agree with results which have been reported previously [2]. In the case of cytochrome u3, the half-reduction potential decreased by 60 mV for each unit that the pH was made more alkaline in the range pH 7.0 to 8.7, but was essentially pH independent in the 6.0 to 7.0 range. Thus, reduction of this component involved addition of one proton per electron to a chemical group with a pK of 7.0, possibly a histidine residue. Cytochrome a exhibited a non-specific pH behaviour, with its half-reduction potential becoming 20-30 mV more negative for each pH unit in the range from 6.0 to 8.7. Arithmetic resolution of the sigmoidal

371

t

Fig. 1. Effect of pH on potentiometric titration of cytochromes a and a3 in submitochondrial particles from pigeon breast muscle mitochondria. Suspensions of submitochondrial particles (2.7 mg/cm’) were maintained under strictly anaerobic conditions in medium containing 0.2 M mannitol, 0.05 M sucrose and either 0.05 M morpholinopropane sulfonate (pH 6.2 and 7.2) or 0.05 M Tris-HCl buffer, pH 8.2. The pH values were checked at the end of each titration. Redox mediators employed were diaminodurene (80 PLM), phenazine methosulfate (80 FM) and ferricyanide (500 PLM). During reductive titrations, diaminodurene was added only after the oxidation-reduction potential was more negative than 290 mV. The measuring wavelength pair was 605-624 nm. Other experimental conditions were as described previously (see Materials and Methods). Both oxidative (A, 0, 0) and reductive (A, n, 0) titrations were performed at pH 6.2 (A, A), 7.2 (0, n) and 8.2 (0, 0) respectively. Sigmoidal titration curves (A) have been resolved arithmetically into the individual components (B). Theoretical curves for an n = 1 acceptor (60 mV per log decade) have been drawn through the experimental points.

titration curves into the contributions of the two constituent cytochromes (lb) also indicated that both species were one-electron acceptors, as evidenced by the slope of the titration curve: a lo-fold change in the ratio of the oxidised to reduced forms was observed for each 60 mV change in the value of U,. Moreover, the constituent cytochromes contributed approximately equally to the overall absorbance at 605 nm, since the point of inflexion of the sigmoidal curves occurred at a ratio of oxidised to reduced heme of unity. The 50 : 50 contribution of the two species is unaffected by changes in pH in the range 6.2 to 9.2. At pH 6.2 and 7.2, cytochrome a3 had a measured half-reduction potential (UI,z) value of 360 + 10 mV, and 300 f 10 mV at pH 8.2 consistent with previous data [2]. The half-reduction potential of cytochrome u was 230 + 10 mV at pH 6.2. 200 + 10 mV at pH 7.2, and 170 k 10 mV at pH 8.2. Spectral changes Measurements of the relative contribution of the two cytochromes to the total alpha-band absorption of cytochrome oxidase have shown that at high levels of azide, the low potential component could account for only about 13% of the overall absorbance change measured at 605 - 624 nm. This presented difficulty in the

372

,

521

550

575

mr,

603

nrr

525

Fig. 2. The reduced-oxidised absorption of the alpha band of cytochrome c oxidase in the presence of 20 mM azide. Submitochondrial particles from pigeon breast muscle were suspended as described in the legend of Fig. 1 at 3.5 mg protein/cm3 and a final pH of 7.2. The dual wavelength spectrophotometer was fitted with a digital scanning attachment which was set to record the absorption spectrum from 510 nm to 640 nm in 30 s using 624 nm as reference wavelength. A baseline (400-400 mv) was established by automatic subtraction of two separately recorded spectra to detect any non-redox contribution to the spectrum. The spectrum (400-228 mv) represents the major (high potential) component reduced in the presence of az.ide. Further reduction to 13 mV (400-13 mv) indicates additional absorption at long wavelength due to the low potential component.

routine analysis of this species over a wide range of experimental conditions. Spectral analyses of the individual cytochromes in the presence of 10 mM azide as shown in Figs. 2 and 3, however, have permitted us to choose a wavelength pair more suitable for measurement of the low potential heme. In Fig. 2 the spectrum of the reduced high potential heme (U,,? = 360 mV at pH 7.2) was the major feature of the alpha-band absorbance, contributing about 88% of the overall absorbance change, with a maximum at 603 nm. Further reduction to an U,, value of 13 mV resulted in a small increase in absorbance with a maximum at longer wavelengths, indicating titration of a component with an absorbance maximum at a wavelength longer than 603-604 nm. Resolution of the low potential species in the presence of azide was achieved by subtraction of two spectra recorded at 219 mV and 19 mV, respectively (Fig. 3). This component exhibited a maximum in the alpha-band region at 610 nm, as compared with 606 nm for the low potential cytochrome in the unliganded state. As cytochromes a and a3 normally contribute equally to the absorbance change at 605 - 624 nm, the effect of azide clearly was to enhance greatly the contribution of the high potential species, while inducing an apparent shift of approximately 4 nm in the absorption maximum of the low potential species to longer wavelengths. Since azide, however, is considered to bind only to oxidised heme, it is probable that this spectral alteration in the low potential species reflects the effects of azide on the half-reduced form of the enzyme. In subsequent experiments, the wavelength pair 610 - 624 nm was employed in order to maximise the contribution of the longer wavelength component to the absorption change in the presence of azide.

313

603 nm

A 1

400

I-D

/

Q

m”-219

m”

0

__^

JOOmV-r400

mV

/

/

h(nm) 525

550

575

600

625

Fig. 3. Absorption changes in the alpha region of the spectrum due to the individual heme components of cytochrome c oxidase in the presence of 20 mM azide. Spectra were obtained as in Figure 2, using a suspension of submitochondrial particles at 3.7 mg/cm3. The component with an absorption maximum at 603 nm (400 mV to 219 mV) represents the high potential species in the presence of azide, with an with an absorption u 1,2,7,2 of 360 mV. Over the range 219 mV to 19 mV, the minor component maximum at 610 nm went reduced as it had an U,,, value of 110 mV under these conditions.

Similar spectral analyses of the Soret absorption bands of cytochrome c oxidase in pigeon breast submitochondrial particles were performed in the absence (Fig. 4) and presence (Fig. 5) of azide. In Fig. 4 spectra were recorded of a fully aerobic sample and after adjustment to 275 mV and 125 mV under anaerobic conditions. From these, the individual spectra of the high and low potential cytochromes were

X(lm)

425

450

/ 475

Fig. 4. Absorption changes in the Soret band of cytochrome c oxidase due to the reduction of cytochrome a and a-,. Spectra were recorded as in Fig. 2 with submitochondrial particles present at 0.6 mg protein/cm3. 50 PM ferricyanide, 20 PM diaminodurene, and 20 PM phenazine methosulfate were added as redox mediators, and 460 nm was used as the reference wavelength. The absorbance change as the sample went from aerobic to 275 mV (anaerobic) represents reduction of cytochrome a3, and that from 175 mV to 125 mV represents principally reduction of cytochrome 0.

314

Aerobc-2GOmV

X(nml 425

450

475

Fig. 5. Effect of azide on the Soret band absorption spectra of cytochromes a and as. Similar conditions were employed for those in Fig. 4 (0.7 mg protein/cm3). Absorbances changes recorded between the aerobic sample and the sample at 200 mV (anaerobic) represent principally reduction of the high potential component in the presence of azide, while primarily the low potential component becomes reduced in the range 200 mV to -20 mV. A baseline was recorded (aerobic-aerobic) by automatic subtraction of two spectra recorded at an interval of approx. 1 min. and a constant V, value.

obtained by subtraction (aerobic - 275 mV and 275 mV - 125 mV, respectively). The Soret maximum for the high potential component occurred at a slightly shorter wavelength, 444 nm, than that for the low potential component (Fig. 4). The relative contributions of the two hemes to the total absorbance of the Soret band were approximately 0.6 and 0.4, respectively. A parallel analysis of the spectra of the high and low potential hemes was performed in the presence of 20 mM azide. In this case, the difference spectra which are presented show fully aerobic to 200 mV (anaerobic) and 200 mV to -20 mv). In the presence of azide (Fig. 5), the absorption spectrum of the reduced high potential component had a split Soret peak with maxima at 443 and 448 nm. The absorbance maximum of the low potential component was apparently unmodified by azide with regard to its position and its fractional contribution to the total absorbance. Oxidation-reduction titrations of cytochromes a and a, absorption band: effect of increasing azide concentration

as measured

in the alpha

A series of titration curves was obtained from experiments conducted at four different azide concentrations, with heme reduction measured using the wavelength pair 610 - 624 nm. The curves, which are shown in Fig. 6, exhibited the same general sigmoidal shape, with the effects of increasing azide concentrations being readily detectable. There was a marked alteration in the fractional absorbance due to the high potential component, as evidenced by a movement of the point of inflection toward lower ratios of oxidised to reduced heme. This represented a change from a value of 0.45 in the absence of azide to a saturating value of 0.67 at

315

rn_i'

O

0

10

Fig. 6. The effect of azide concentration on the course of oxidation-reduction of cytochromes a and aa at pH 6.2. Titrations were conducted as described in the legend of Fig. 1 except that an additional mediator, 5 x 1O-5 M FeSO, + 3 mM EDTA, was also included. Oxidative (0, A, 0, 0) and reductive (0, A, +, n) titrations are represented at 0 (0, l), 0.08 (A, A), 0.8 (0, +), and 8.0 (0, n) mM azide, respectively. Each sigmoidal curve (6A) has been separated into its individual components (6B) and the U,,* value of each species noted.

high levels of the inhibitor. Azide concentrations of 0, 0.08, 0.8 and 8.0 mM were chosen for the titrations shown in Fig. 6 because they correspond to 0, 1,lOand 100 times the apparent dissociation constant, which could be inferred from half saturation of the change in relative fractional absorbance and from the changes in the observed U, ,2 value of the low potential component at pH 6.2. In the latter case the apparent dissociation constant for binding of azide to the oxidised form was calculated using the equation:

u1,2 =

U, + 0.06 log

[~31)K7 (fG + W3l)K

(4

+

where K, and K, are the dissociation constants for interactions with oxidised and reduced cytochrome, respectively, and U, is its half-reduction potential in the absence of azide. At a concentration equal to the dissociation constant K,, azide should cause a decrease in the midpoint potential of this component by 18 mV. Thereafter, for each tenfold increase in azide concentration, the resultant lowering of the midpoint potential rapidly attains a value of approximately 60 mV per decade, assuming K, is large and providing the stoichiometry of binding is unity [17]. This is in agreement with the observed behaviour. In three experiments, the midpoint potentials at pH 6.2 were 230 mV t_ 10 mV at zero azide, 210 mV f 10 mV at 0.08 mM azide, 160 mV f 10 mV at 0.8 mM azide, and 110 mV f 10 mV at 8.0 mM azide. A value of 0.25 mM for the apparent K, at pH 7.2 has been reported previously, using similar techniques [2]. In contrast, the half-reduction potential of

376

[OXI

L0q [Red] -1 0

0

10

Fig. 7. Effect of pH on the half-reduction potential of the high potential component of cytochrome c oxidase in the presence of 20 mA4 azide. Suspensions of submitochondrial particles from pigeon breast muscle (2.9 mg protein/cm3) were titrated under standard conditions (Figs. 1 and 6) at pH 6.2 (0, l), after reduction of the 7.2 (A, A), 8.2 (0, W), and 9.2 (0, +). I n each case, the titration was terminated high potential component only (approx. 200 mV) and this value taken as 100% reduction. Oxidative (0, A, 0, 0) and reductive (0. A, W, +) titrations were conducted at each pH value).

the high potential species added ligand concentration. pH dependencies

appeared

to be essentially

of the individual hemes of cytochrome

independent

(+ 10 mV) of

oxidase in the presence of azide

The pH dependencies of the two components were also examined in potentiometric titrations in the presence of 20 mM azide. The high potential component (Fig. 7) exhibited an approx. 30-50 mV decrease in lJr,2 per pH unit over this range which could not be readily distinguished from the pH behaviour of unliganded cytochrome

Fig. 8. Effect of pH on the half-reduction potential of the low potential component of cytochrome c oxidase in the presence of 20 mM azide. Standard titration conditions were employed (Fig. 6) with particles at 3-4 mg protein/cm3. Open symbols (0, A, 0) and closed symbols (0, A, n) represent oxidative and reductive titrations, respectively at pH 7.2 (0, l), 8.2 (A, A), and 9.2 (0, n).

a3 (Fig. l), except in the pH range 6.0-7.0. The alteration in the U1,z of this species was slightly more pronounced at more alkaline pH values. In all cases, the slope of the curve as a function of U,, approximated to that of a one electron acceptor. Measurement of the pH dependence of the low potential component at 20 mM azide (Fig. 8) demonstrated clearly that the Ur,* value of this species remained equal to 93 + 10 mV in the pH range 7.0 to 9.0. Assuming that the active inhibitory species was the undissociated acid, HN,, pK = 4.6 [18], this apparent pH independence (at constant total azide) may represent a situation in which the effect of decreasing HN, at more alkaline pH was exactly counter-balanced by the intrinsic low potential species. DepH dependence of the lJ1,2 value for the unliganded creased HN, concentrations would result in a shift of the Ul,2 value to more positive potentials, while more alkaline conditions would lead to more negative midpoint potentials of both cytochromes a and u3. DISCUSSION

In this communication, we have extended an earlier study on azide interaction with cytochrome c o.xidase [2] and further demonstrated the effects of this inhibitor on the characteristic spectral and thermodynamic properties of both cytochromes a and u3. The former include detailed examinations of azide-induced spectral effects on cytochromes a and u3 in the alpha and Soret band regions. The thermodynamic studies include a comprehensive analysis of the variation in the midpoint potentials of the visible components with azide concentration, coupled with measurements of their pH dependencies in the presence of ligand. The correlation of these changes with the known intrinsic pH dependencies of cytochromes u and u3 has allowed us to draw inferences concerning the site and the mechanism of action of the azide ligand. The complexity of the induced effects and the absence of any simple pattern of comparison with the properties of the unliganded hemes have precluded an unequivocal identification of the azide binding site. Spectral alterations

induced by adding uzide to cytochrome

c oxiduse

The principal effect of the azide on the measured absorption band of reduced cytochrome oxidase was to promote an increase in the extinction coefficient of the high potential component by greater than 50% with no apparent change in the wavelength of its absorption maximum, which remained at 6033604 nm. This phenomenon could be observed directly by spectral analysis (Fig. 3) or in titration curves by the movement of the point of inflection to lower apparent ratios of oxidised/reduced heme with increasing azide concentration (Fig. 6). This ratio attained a limiting value of 0.67 using the wavelength pair 610 - 624 nm. Simultaneously there was an apparent shift in the low potential component (relative to the intermediate azide-liganded state) to longer wavelengths with a maximum at 610 nm, as compared with 606 nm for this species (cytochrome a) in the absence of azide. Changes due to other ligands in cytochrome oxidase, such as addition of CO

378

or those induced by ATP addition [19], also have included alterations in the relative interaction. absorbances of both cytochrome a and a3, in part through heme-heme Indeed, the spectral changes induced in the oxidised form of cytochrome oxidase by ATP are very similar to those described for cyanide [20]. Previous studies of cytochrome oxidase at liquid nitrogen temperature [21,22] indicated that cytochrome a3 had a symmetrical peak at 444 nm while cytochrome a exhibited a unique double maximum at 442 nm and 448 nm. This unusual split peak was observed in a variety of experimental situations including the aerobic steady state in the presence of H,S, HCN, HN,, or CO, and was considered to be characteristic of cytochrome a. As shown in Fig. 4, the percentage contributions of a3 and a to the reduced Soret peak were approximately 60 : 40 at 445 nm - 460 nm with maxima occurring at 444 nm and 446 nm for a3 and a, respectively. For samples at room temperature, no double peak was observed for cytochrome a, although this peak was considerably broader than that of cytochrome u3. In the presence of azide, a double Soret maximum was observed for the high potential component even at room temperature, indicating that the heme was in a highly asymmetric environment. Cytochrome a, the higher potential component in a suspension of intact mitochondria in the presence of ATP, exhibited a similar Soret band. Thus the spectral evidence for azide binding indicates preferential interaction of this inhibitor with the oxidised form of cytochrome u3, with spectral effects similar to those previously described for addition of ATP to well coupled mitchondria. Azide-induced effects on the oxidation-reduction cies of cytochromes a and a,

midpoint potential

and pH dependen-

In contrast to the spectral information, the potentiometric data for azide binding to cytochrome c oxidase suggested, at least in the simplest interpretation, a direct interaction of this inhibitor with the oxidised form of cytochrome a [2]. This interaction was manisfested by a decrease in the midpoint potential of the low potential component with increasing azide concentration, a dependence which rapidly approached 60 mV per lo-fold increase as the azide levels were raised above the apparent K,. This behaviour would be expected for preferential binding of a ligand to the oxidised species of an oxidation-reduction component in a 1 : 1 stoichiometric relationship [23]. In this connection, no azide concentration dependence of the U,,,2 value of the high potential titrant was observed, despite alterations in the spectral properties of this component. Analysis of the pH dependencies of the U1,2 values of the high and low potential species in the presence of constant concentrations of azide, however, revealed that the latter differed markedly from those observed for cytochrome a in the absence of inhibitor while only minimal alterations were observed for the high potential species (corresponding to cytochrome us in the unliganded state). Thus the U,,* value of the low potential component at high levels of azide became essentially independent of pH, in contrast to the 20-30 mV dependence per pH unit of cytochrome a.

319

Does azide bind to oxidised cytochrome

a or a,?

In view of the complex spectral, I!J~,~ and pH dependence alterations induced by azide, mediated uia heme-heme interactions, it becomes difficult to define precisely the primary binding site for this inhibitor. However, all the major models of cytochrome oxidase consider the a3 heme to be the terminal oxidase which reacts directly with oxygen and a variety of ligands including CO, HN,, HCN and NO. Thus, in this case, it is necessary to account for the surprising observations, as revealed by potentiometric titrations, that azide appears to interact directly with cytochrome a, since it is the low potential species with exhibits a -60 mV dependence/tenfold increase in azide concentration, indicating preferential binding of the ligand to the oxidised cytochrome. The two main views of cytochrome c oxidase which have been put forward are: (a) the two-cytochrome hypothesis emphasising the individuality of cytochromes a and a3 as orginated by Keilin and Hartree [24] and (b) the single cytochrome or special dimer hypothesis which envisages the two cytochromes as identical species, interacting to the extent that the properties of each are determined by random selection of which is reduced first (see Ref. 25 for review). In recent years, it has become apparent from all measured parameters that the 2 hemes are non-identical in the fully oxidised or reduced states of the enzyme. However, it is equally clear that heme-heme interactions occur such that the spectral and thermodynamic properties of the a and a3 hemes are influenced by the oxidation/reduction state or the presence of ligands on the companion heme. Wikstrom et al. [26,27] have suggested a modified special dimer model in which negative redox interactions between two hemes with identical half-reduction potential gives rise to the observed biphasic redox behavior. In this scheme, an electron would have an equal probability of reducing a of a3 in the oxidised enzyme. Thereafter, the negative cooperativity would dramatically reduce the affinity of the remaining heme for a second electron, resulting in a decrease in Ui,2 value of approx. 160 mV. Thus, each arm of the sigmoidal titration represents a random mixture of a and a, hemes titrating in parallel as high and low potential forms. The special dimer model, even with two different cytochromes, appears inconsistent with much of the available data. The hemes of cytochromes a and a3 are physically quite widely separated in the protein (see for example Oh&hi et al. [28]) and the required interaction energy of approximately 3.5 kcal/mole would have to be transmitted by protein conformation changes. More importantly the hemes appear to be in very different environments and this is difficult to reconcile with their having identical half-reduction potentials. The high potential copper atom, for example, is an integral part of the environment of cytochrome a3 and the oxygen reaction site [9,10,13,14,29] while the low potential copper is not closely associated with cytochrome a (,see for example Ref 28). Attempts to directly measure the effect of the redox state of cytochrome a3 on the half-reduction potential of cytochrome a give values of 40-60 mV [21,22], far less than the 160 mV required by the special

dimer model. Indeed in our hands, cytochrome a always exhibits a characteristic double Soret maximum whether cytochrome a3 is oxidised/reduced or bound to ligands such as HCN, CO or azide [21,22]. Against this background, the a priori evidence presented in this communication would indicate that azide binds directly to cytochrome ~1. This interpretation is not certain, however, because heme-heme interaction prevents unambiguous identification of the a and a3 hemes in the liganded state. Additionally, the spectral and pH behaviour of the two heme components as described in this communication are consistent with the primary interaction of azide with the oxidised form of cytochrome u3. Thus the high-potential species in the presence of azide, exhibits a double absorbance maximum in the Soret region, normally considered to be characteristic of cytochrome a. Similarly, the apparent pH independence of the low potential species in the presence of 20 mM azide may be attributable to a 60 mV/pH unit dependence on the change in HN, concentration, matched by an equal and opposite intrinsic pH dependence [60 mV/pH unit) of this species, characteristic of cytochrome ug in the unliganded state. The switch in U1,2 values so that cytochrome a3 becomes the low potential component in the presence of azide is possible if the inhibitor binds with high affinity to oxidised a3 in the minor half-reduced species a3 .3+. Cu'+.a’+. This would stabilise this minor intermediate [30], the formation of which would be thermodynamically unfavourable in the absence of ligands. A detailed analysis of the possible mechanisms and their relative strengths and weaknesses is presented in the accompanying paper. AKNOWLEDGEMENTS

This work was largely carried out in the U.S.A. and was supported by Grant GM12202 from the U.S. National Institutes of Health. C.S.O. is the recipient of USPHS Research Career Development Award GM00318 from the National Institutes of Health. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12

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