Eur J Biochem 120, 339-344 (1981) < FEBS 1981

Electron Transfer between Azurin from AZcaZigenes faecalis and Cytochrome cs51 from Pseudomonas ueruginosu

Philip ROSEN, Michacl SEGAL, and Israel PECHT

Department of Chemical Immunology, The Weizmann Ihstitute of Sciencc, Rchovot

(Rcceived April 21 ;July 22. 1981)

The electron transfer equilibrium and kinetics between azurin from Alruli~enr.s,fueru/i.s and cytochrome r ~ s I

from Pseudomonas aeruginosa have been studied. The equilibrium constant K = { [Cyt(III)] . [Az(I)]) I . ( [Cyt(Il)] . [Az(IIj]) = 0.5 at 25°C is about seven times smaller than that observed between the cytochrome c5s1 and the azurin both from P. ae~ugizzosa [Rosen, P. and Pecht, 1. (1 976) Bioclzemisf~y, 15, 775 - 7861. Potentiometric titrations confirmed a 43-mV difference between the mid-point potentials of + 266 mV and + 309 m V for the Alculigenes and Pseudoinonas azurins respeclively .

The kinetics of the reaction between Akaligenc~s azurin and Pseudomonas r~torlz~oznc cSs1 were investigated by the temperature-jump chemical relaxation method. Only a single relaxation mode was observed throughout the range of concentrations and temperatures examined. Thus, the slow relaxation time observed in the reaction betwecn P. acwginosu azurin and cytochrome cSs1 is not observed with the AIc~l igezz~ .~ azurin. The simplest mechanism that can therefore be ascribed to the investigated system is:

Cyt(I1) + Az(I1) Cyt(TI1) + Az(Ij \lr

Cyt ( I I IT. This scheme is similar to that proposed earlier for the reaction between P. ucmginosa azurin and cytochrome ~ ' 5 5 ~

but does not involve the conformational transition proposed for azurin. The specific rates for the elcctron transfcr are still fast: 1.8 x 10' M-' . s - ' and 3.0 x lo6 M-' . s - ' respectively at 25 'C.

Thc electron-mediating proteins, cytochroine r55, and the blue copper protein azurin, can be obtained from the cell extracts of many specics of Pseudonzonas and Akuligene.7 [l ,2]. The proteins are small, readily isolated, and their redox changes are easily monitored. They have been shown to be components of the bacterial terminal respiratory network that can use either oxygen or nitrate as the terminal electron acceptor [3].

The electron transfer between the Pseudomom7 cyto- chrome rsS1 and azurin has been studied extensively [4--81. All the reports so far have mentioned the remarkably high electron transfer ratcs between these proteins. Values of IO ' - lO7 M- ' s at 25°C for the electron transfer were measured, which decreased by about lo4 when horse-heart cytochrome c was substituted for the Pseuclon~ona~s cyt r551 [4]. The chemical relaxation spectrum of the temperature- jump perturbed equilibrium between azurin and catochromc ('551 (both from Ps. u~rugiiiosa) revealed two relaxation modes. These were intcrpreted in terms of a three-step reaction scheme involving conforinational equilibria between reactive and unreactive conformers of the reduced azurin and of the oxidized cytochromc ('551, that modulated the electron transfer equilibrium [7]. The activation parameters for the elcctron traiisfer showed large positive AS' and A H + , in contrast to the generally small values of AH' and negative AS' that

Ahbrrviuziorts. Cyt(1l) and Cyt(JlI), rcduced and oxidized cylo- chrome c551, rcspcctivelp; Az(1) and Az(II), reduced and oxidixd azurin, rcqmtively.

have been observed when one of the proteins was substituted by a low-molecular-weight redox reagent 191. These differences were therefore regarded as being indicative of 'specific intcr- actions' between the proteins originating from areas of com- plementary charge on the two proteins that exist at neutral pH. The recent Marcus theory analysis of the reactions between r-type cytochromes and blue copper proteins has however, suggested that, in spite of the high reactivity ob- served for some of these couples, no kinetic selectivity is found in their reactions [8].

The tertiary structure of both Pseudomonas cytochrome ~ ' ~ 5 ~ and azurin have recently been solved. Although the scquence of the first has large deletions, in comparison with that of the horse-heart cytochrome c, it still retains the overall folding pattern of the eukaryotic cytochrome r [lo, 111. The shortened chain results in the heme being more exposed and this could probably be a inajor reason for the very high clcc- tron self-exchange rate that has been found [7,8,12]. and for its much higher rate of reaction with azurin in comparison with the latter's reaction with horse-heart cytochromc r. The structure of Pseudomorias azurin has recently been determined and refined to 0.27 nm [13,14]. The coordination site of the copper ion has been shown to involve two imidazoles ( I Iis-46 and His-117), a thiolate (Cys-122) and a methionine Sulphur (Met-121). The distorted tetrahedral symmetry and the chemical nature of the ligand atoms confer on the copper ions their unique reactivity and spectroscopic properties [I 5 - 171. Detailed features of the surface groups of Psrudomorztr.s azurin and spatial relationship between the Cu site and poten-

340

tial electron transfer loci were deduced from that three- dimensional model [I41 and from affinity labeling experiments with Cr(I1) ions [18].

Alcaligenes faecalis is also a member of the large and varied group of nitrate-reducing bacteria. Although its azurin differs from that of Ps. aeruginosa azurin in a third of its amino-acid residues [2], the changes are for the most part conservative ones. The net result is an increase in the number of charged residues at the expense of the hydrophobic ones. Major sequence differences which may be of functional significance are at the neighborhood of His-35 of Alcaligenes azurin. Thus, Pro-36 and Ser-34 of Pseudomonas azurin are substituted by a threonine and lysine respectively [2]. Such substitutions may cause marked conformational differences around that irnidazole. In our study of the reaction mecha- nism between Pseudomonas azurin and cytochrome ~ 5 5 1 ,

two conformational equilibria were proposed ; a fast one (z 2 0.1 ms) in the oxidized cytochrome c j S 1 , and a slow one (z z 40 ms) in the azurin [7]. More recently we have ob- tained evidence that the conformational equilibrium of Pseu- domonas azurin may be observed also in the absence of cyto- chrome c551 and is a proton transfer coupled process [29] (Wherland and Pecht, unpublished). In our affinity labeling study of azurin, we have shown that this particular area on the surface of Pseudomonas azurin acts as an electron transfer site with imidazole-35 being part of a potential elec- tron relay system from the protein-solvent interface to the copper [181. All these findings made the examination of the reaction between Alcaligenes azurin and Pseudomonas cyto- chrome cSs1 worthwile. The study of the cross reaction be- tween proteins of different species with defined structural differences may provide us with an assignment of the site of conformational transition in Pseudomonas azurin.

MATERIALS AND METHODS Preparation of Proteins

The bacteria from which the proteins were extracted were Pseudomonas aeruginosa and Alca1igene.s fuecalis, generously supplied by Dr R. P. Ambler. Both were grown without aer- ation in a 450-1 fermentor. The growth medium, the conditions of growth, harvesting and storage of the bacterial paste, were as previously described [7]. The isolation of the Pseudomonas cytochrome c551 and azurin followed the procedures of Am- bler [I, 19,201 but the preparation of the AlculigenPs azurin included small changes from Ambler's procedure for purifi- cation of Pseudomonas azurin. First, the initial adsorption onto the CM-cellulose (CM23) was at pH 4.05. Second, during the fine separation on CM-cellulose (step 3 of Ambler's 1963 procedure [19]) the Alcaligenes azurin was eluted at pH 4.9 instead of pH 4.65 used for the Pseudomonas azurin. Although several cytochrome bands were eluted from the column below pH 4.9, the amounts were all small. No cytochrome remained on the column after the Alcaligenes azurin had been eluted. The ammonium sulphate step had to be aban- doned as no precipitate could be obtained under the con- ditions used for the Pseudomonas azurin [l].

The yield of Alcaligenes azurin was very large, 450 mg/kg of cell paste, compared to 120 mg/kg of cell paste for the Pseudomonas azurin. The absorption ratios for the three pro- teins were Pseudomonas cytochrome ('551, $2; /Ez;O = 1.16: Pseudoomonas azurin, ~ ~ , " / c ~ ~ ~ = 0.52; Alcaligenes azurin, ~,6x"'/p,',"~ = 0.50. All proteins were stored at 4°C in 0.05 M ammonium acetate buffer at pH 3.9.

Reagents Platinum black, obtained from Fluka (Buchs, Switzerland),

was washed with 10% nitric acid and distilled water, and dried under vacuum before use. Methyl viologen was obtained from BDH and proflavine hemisulphate from Sigma. 1,iC-Ben- zoquinone, used as mediator in the pentiometric titrations, was purified by boiling with activated charcoal and twice recrystallized from aqueous alcohol (50% v/v). All other chemicals used were of analytical grade. The buffer used was potassium phosphate ( I = 0.1 M) at pH 7.0 [21], and in- cluded 0.01 mM EDTA.

Procedures The reduction of the Pseudomonas cytochrome e5j1 by

hydrogen, catalyzed by platinum black, and the static titra- tions were conducted as previously described [7]. The ab- sorption coefficient at 625 nm of the oxidized Alcaligenes azurin was determined by titrating the reduced protein with potassium ferricyanide and found to be 4000 100 M-' cm-' compared to 5700 & 100 M-' cm-' for the Pseudomonas azurin using the same procedure [ 7 ] .

The kinetic titrations were carried out in a double-beam temperature-jump apparatus [22] using a cell modified for anaerobic work (Pecht, I., unpublished). The procedure used has been described elsewhere [7]. Temperature jumps of 2.9 "C were used for all measurements. The spectral changes were monitored at 551 nm and the relaxation spectra were recorded in a Biomation 802 transient recorder. The relaxation times were evaluated using the algorithm of Strehlow and Jen [23] on an H P 2100 computer.

The potentiometric titrations were done in a Thunberg cell modified according to Reinhammar [24]. A silver/silver chloride platinum electrode, 3 M KC1, was used for the mea- surements. The reference potential was + 208 mV.

RESULTS

Siatic Tiirations

The values of the equilibrium constant K = [Cyt(III)] [Az(I)]/ [Cyt(Il)] [Az(Il)] obtained by titrating different start- ing concentrations of reduced Pseudonzonas cytochrome c55 1

by oxidized Alcaligenes azurin at various temperatures are shown in Table 1. It can be seen that there is a very slight increase in K with temperature, i.e. that the formation of oxidized cytochrome ('551 is mildly endothermic. A van't Hoff plot gave a value of + 2.38 kJ . mol-' for AH,,,. Interestingly, the overall equilibrium constant for the Pseudomonas azurin/ cytochrome csS1 system [7] was also found to be independent of temperature. The value of the latter (3.9), defined and obtained in the same way, is a factor of seven larger and means that the mid-point potential of the Pseudomonas azurin is some 40 mV higher than that of the Alcaligenes azurin.

Potentiometric Titrutions

In view of the large difference between the mid-point potentials (EO') of the azurins revealed by the static titrations, direct measurements of them were carried out, and the resultant Nernst plots are shown in Fig. 1. As can be seen, the slopes of the lines are close to unity (0.95 and 1.02 for the Alcaligenes and Pseudomonas azurins, respectively), as ex- pected for a single electron transfer. There is a difference of

Table 1. The overall equilibrium consmnf ,for the mixed Alcaligenes/ Pseudomonas sysrem at dfferent concentrations of reduced cytochrome c551

and ai various temperatures -

[Cyt css*(II)I Temperature K,,,

c

Fig. 2. Plors of ihe reciprocal rela.xulion iinzes at 35' C of rhc Alculigenes nzuririiPseudomonas cytochrome c551 sysiem f o r rhree siuriing mnc'en- rraiions of rhe cyrochrume. Total concentrations of cytochrome ('551 were (A) 13 pM, (0) 33 pM, (0) 66 pM. Insert: chemical relaxation of the Alcaligenes system after a temperature-jump of 2.9 T, monitored at 551 nm and showing net decrease in transmittance. Final temperature was 25 'C. Oscilloscope settings: 1 ms/division, 0.33 '%, transmittance/division. Total concentrations: Pseudomonas cytochrome cs5l 66 p M ; Alculigenes azurin, 150 pM

PM

33.2 30.6 64.6 67.5

~~

" C

5 0.508 25 0.568 25 0.541 35 0.562

I -1.0 -0.5 0 0.5 1 .o

log b z ( I I ) l l b z ( I ) l

2001 /i, I s , I I , ' I I " ' I I " ' I " I

Fig. 1. Nernsi p1oi.r o f rhr poirniiomeiric ijirarjons oJ Pseudomonas (0) and Alcaligenes (0) azurins. Potassium phosphate buffer, I = 0.1 M, p H 7.0 at 25 'T. Mediator was 1,4-benzoquinone at 15 pM

43 mV between the two i?" values. These were + 266 mV and + 309 mV for the Alcaligenes and Pseudomonas azurins, respectively. The latter value approximates to the + 304 mV obtained by Goldberg and Pecht [9].

Kinetic Titrutions

These titrations involved the addition of oxidized Alcali- genes azurin to reduced Pseudomoms cytochrome ('551 over a wide range of temperatures and concentrations of both components and subjecting them to a temperature-jump perturbation [25]. The system showed a single relaxation mode only (Fig.2 insert). The plots of T - ' against the sum of the equilibrium concentrations of reduced cytochrome c551

and oxidized azurin shown in Fig.2, were all measured at 35°C. Each of them represents a titration of oxidized azurin against a fixed initial concentration of cytochrome ('551. The same kind of titrations are shown in Fig. 3, but here tempera- ture is varied while the initial cyctochrome ~ 5 5 1 concentra- tion is the same for the four curves. It can be seen from Fig. 3 that by increasing the starting concentrations of cyto- chrome c551 the values of the intercept increase; from Fig. 2 and 3 it is seen that, at low concentrations, the relaxation times deviate significantly from the straight line obtained at the higher concentrations, tending to converge towards the origin. Further, the value of the reciprocal relaxation time did not level off at high azurin concentrations but increased linearly with it.

As only a single relaxation mode was observed the simplest mechanism that can be proposed is a one-step bimolecular process :

Cyt(I1) + Az(ll) 1'11 Cyt(II1) + Az(1) (1)

3.2

15'C

0 ==?F, ; , ; , , - , , , , , , , , , , 0 40 80 1 2 0 1 6 0 200 240 280 320 360

where the electron transfer is assumed to occur within the encounter complex, the lifetime of which is very short com- pared to the average time required for the electron transfcr. Thus, the observed relaxation time is due to the electron trans- fer equilibrium. However, if that was the only equilibrium pres-

ent in this system, then one should expect a rather small relaxa- tion amplitude, if any at all, in view of the above finding of AH,,,, = 0. Hence the observed relaxation implies the presence of a further step. Indeed the Pseudomonas cytochrome c551 has been suggested to undergo an isomerization reaction [7] and is therefore expect to exhibit this step here, being the common reactant in the present system and in that where it reacts with the Pseudoomonus azurin.

The difference between the Pseud(monu.7 system and the present one is that no transition due to the azurin is observed. The simplest scheme would now evolve to the following:

Cyt(I1) + Az(II), ' I ' i l l ' C ' > ~ t ( l I I ) + Az(I).

/%, ,11 , (2) Cyt( I I I )*

The relaxation time would therefore show (cf. Appendix) the following dependence on the concentration of reactants:

F 1 = k12 ([Cyt(II)] + [Az(lI)]) + kzi ([Cyt(lII)] + [Az(Q]/(l + KO)} ( 3 )

where Ks = [Cyt(III)*]/[Cyt(III)]. Concentrations were varied by adding Az(I1) to Cyt(I1) and, under our experimental conditions were oxidized azurin is in excess. we may ap- roximate :

16.8.

(4)

where [CytItot is the total amount of cytochrome L' present and by stoichiometry we may approximate that

[A$)] = [Cyt(III)] + [Cyt(III)*] [Cyt],,,. ( 5 ) The value obtained for KO in our previous study [7] should also be applicable in the present system as it should not depend on the type of azurin employed. This allows one to calculate k21 while k12 was determined froin the slopes of Fig. 2 and 3 . These data are summarized in Table 2.

Thc Eyring plots of kl2 and k21 yielded the following parameters for the electron transfer processes: A H!2 = + 75.7 kJ . mol-' and A H & = + 63.5 kJ . mol-' and A S 1 2 = + 129.6 J . mol-' . K- ' and A S $ = + 94.0 J . mol-' . K-'. The dif- ference between the activation enthalpies shows that the formation of oxidized cytochrome cSs1 is an exothermic pro- cess ( A H = - 12.1 kJ . mol-') and yet it is this direction in which the reaction is found to be driven on raising the tem- perature (cf. Fig.2 insert). This is indeed further evidence for the involvement of an extra endothermic step in that system, which we assume to be the cytochrome ('551 isomer- ization.

The amplitude data obtained for this system may be analysed in terms of a simpler expression than that em- ployed in the earlier scheme of the Pseudoornonus system as only two steps are involved here (cf. [7]):

r-' = [Cyt(lI)]-' + [Az(II)]-' + [Az(I)]-' + l /[cyt(rrr)] . (1 + KO). (6)

The expression for the amplitude will become:

where A I = change in light transmission (in V), lo = total light signal (in V), A T = rise in temperature (= 2.9 K), T = absolute temperature, R = the gas constant, d = light path of the temperature-jump cell (= 0.7 cm), Ae, = net

Table 2. Elecrroti iransfer rcire consfants ar different ternperaiures

Tempcrature lo-' x Ratc constant ~ .~ ~ -~ ~ -~ ~- ~. ~~

lClZ kzi

'C M-1 s - I

~. - - ~ ~~

15 0.81 1.5 25 1.8 3.0 35 5.8 9.2 45 13.7 15.1

AH+,+=2.31kJ rnol-' /

0 2.0 4.0 6.0 8.r 0

~ .-

r (wrnol)

Fig. 4. Plot of die anzp1itude.r of dw relrix-arioi~ niodcJ according to Eqn (8) . Total concentration of Pseudonionu.s cytochrome C S ~ ~ has 66 pM. r = 3 5 c

change in absorption coefficients for the electron exchange equilibrium of Eqn (1) (= 1.7 x lo4 M - ' cm-' at 551 nm).

This equation can be rearranged so as to allow plotting of the experimental data:

(8) KO

1 + KO Fig.4 shows the resulting plot which yields A H = 2.31 kJ . mol-'. This value falls into the range observed by other measureinents of the temperature dependence of the overall equilibrium of that system.

A H = A H , + - - . A H O .

DISCUSSION

The marked difference between the redox potentials of Psc~udonionus and A1culigene.s azurin finds at least its partial rationale upon comparing the sequences of these two proteins. The copper-coordinating residues are conserved in all azurins (and homologously in plastocyanins) sequenced to date [2]. Hence one can safely assume that the ligand sphere of the metal ion is identical in the two azurins. The change in potential may then be caused by (a) different geometry of thc ligand sphere or (b) environmental differences such as in the internal protein dielectric, or (c) a combination of both previous changes. One is inclined to give less weight to the

343

first possibility on the grounds that the site symmetry has apparently evolved so a s to minimize the Franck-Condon barrier. Therefore minimal variation in coordination geometry would be expected between these closely related proteins. Thus, the likely cause for the difference is environmental. Indeed one finds [2] that neighboring residues of the coordi- nating side chains are very different in the two proteins: the stretch between His-I 17 and Met-121 consists of Ser-Ala-Leu in Psru~~oion.ronus azurin and this is substituted by Trp-Ser-Ile in the protein. The highly conserved lysine following Met-1 21 in tlie former is a threonine in the latter. Also the single, highly conserved Trp-48, which lies close to the copper site in P.seiidomorzn.s azurin, is subs t i t u ted in A kaligmes azurin by a valinc. These extensive differences in primary structure are bound to cause changes in the medium in which the site is embedded. The difference in redox potential is hence a reflection of these changcs.

The conformational transition proposed to occur in P.scudonzotiu.r azurin and to modulate the electron transfer process is absent when the Alcaligcrzes azurin is the electron transfer partner. This finding now gains special significance in view of several recent advances.

High-resolution NMR studies of P.srziclonionu.r azurin have shown tlie presence of a non-copper-coordinated imid- azole residue in this protein with an unusual proton exchange rate [26-281. More recently comparison of the NMR data and the three-dimensional structure [I 81 as well as direct studies by Mitra and Bersohn (personal communication, 1981) provided evidence tha t this is the imidazole of His-35. This point is amplified below. Silvcstrini el al. [29] have very re- cently investigated the pH dependence of the electron transfer kinetics between Pscudomonus azurin and cytochrome c S J 1 .

Their results confirm the general scheme proposed earlier involving a pH-independent electron transfer step and a pH- dependent slow transition between two conformers of rc- duced azurin. The pK of this transition was found to be about 7.0 and was therefore assigned to a protonation equilibrium of one of the non-coordinated imidazoles [29]. Thesc observa- tions are in accord with our proposal that tlie slow proton exchange of His-35 should be rclated to tlie slow transition between the active and inactive conformer of azurin [I 81.

By employing q u o Cr(I1) as an affinity labeling agent, an electron transfer locus defined by the peptide loop Lys-85 to GIu-93 on P.seudonzoiius azurin has been identified [I 81. The electron was proposed to proceed through a n orbital relay systcin composed of the imidazoles of His-35 and His-46. That this locus is of functional significance has been shown by the relative attenuation in reactivity of the Cr(ll1j-labeled Psrudoionzonas azurin with Pseudonzonas cytochrome c 5 5 1 and the absence of any such effect on the reaction with Pscudo- ~170nu.s c.ytochronzc oxiduse (0. Farver, Y. Blatt and 1. Pecht, unpublished results). These results implied that: (a) the Cr(II1)-labeled site on azurin is the one reactive with the cytochrome (551 and that (b) the Pswdoomonas azurin has at least onc further active site for reacting with the oxidasc and (c) that His-35 is directly involvcd in the electron transfer process.

The results of the present study clcarly show thc impact of the sequence differences between Psruclomoizas and Alculi- ~ P W P , Y azurins, in the immediate neighborhood of His-35, on their reaction patterns. As mentioned above, Ser-34 and Pro-36 in the former protein are substituted by a lysine and a tlireo- nine rcspectively. Although n o three-dimensional structural information is yet available for Alculigencs azurin, such sub- stitutions should obviously cause a pronounced structural

diff'erence around His-35. Examination of the structure of Pscudonzoizus azurin [I 3,141 shows thc marked bend caused by Pro-36 in the proximity of His-35. Its substitution by a thrco- nine in Alcaligenes azurin may drastically alter the solvent accessibility and hencc reactivity of that imidazolc. It is therefore not surprising that the protonation-dependent con- fbrmational transition of Pseudomonus azurin absent in the reaction of Alculigenrs azurin with the common Pseurlotnorius cytochrome ('551 partner. This could suggest that His-35 no longer plays a role in the electron transfer. However in view of the recent PMR studies of Mitra and Bersohn (private communication) and the observed rates of electron transfer which are only slightly attenuated in the latter reaction one may suggest the operation of a common electron relay systcm even though the activation parameters are again quite dif- ferent, particularly for the azurin oxidation. We favor the latter possibility but resolution of this problem requires apply- ing tlie Cr(1l) affinity labeling procedure [I81 to AIui1igerzc.s azurin and examination of the locus and reactivity of the Cr(II1) product along with a high-resolution crystal struc- ture of this protein.

In conclusion, the results of this study of the electron transfer cross reaction between Alcaligcwes azurin and P.srudo- nzotzus cytochrome cS5, have several revealing aspects. (a) The :tssignment of the slow monomolecular step obscrved in the Psmdonzonas azurin/cytochrome c551 reaction to a conforma- tional transition in the azurin is further substantiated; so is the mechanistic scheme which involves a transition between two conformers of cytochrnme ('551. (bj The differences in sequence between the two azurins are expressed in a marked difference in the redox potential of the two proteins, yet have a relatively small effect on the actual electron transfer rates with cytochrome cS5,. (c) The rather large activation enthal- pies and positive entropies that characterize the electron trans- fer steps of Alculig~ne,~ azurin relative to P.cctidonioizci.c azurin reflect possible differences in the potential energy surface preceding actual electron transfer. (d) Thc proposed special role of imidazole-35 in an electron rclay to the copper of Pscudoomonus azurin [I 81 received independent corroboration.

APPENDIX

The relationship of the affinity to the reaction advancement for each step and the coupling between equilibria is expressed in the diagonal and off-diagonal terms, respectively, of the g matrix as shown by Castellan [31]. For the scheme of Eqn (2) it is given by: (see p. 344).

The authors wish to thank Drs M. Goldberg. Y . Blatt and P. F r a n k for thcir valuable advice and cornmcnts during the preparation of this paper. We are very grateful to Dr R. P. Ambler for supplying us tlie strain 01' A/cu/i,ycwes faeccrli.i and lo Drs R. Rersohn. M. Brunori and E . T. A d m a n for corninunicxting to us unpuhlishcd r~st i l ts .

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of Prorciti S i i u c ~ r i i i - c ~ , c (Prcviero, A,, Pecherc. J.-F. "+ Colclti-Prc- viero, M.-A., eds) pp. 289-305, Inscrm, Paris.

3. Horio, T. (1958) J . Bi~xIre17~. (Tokyo) 4.5, 195-205. 4. Antonini, l!.- Finnazi-Agro, A, , Avigliano, L . , Guerrieri, I> . , Rotilio,

5. Pccht. I. CYL Rosen. P. (1 973) Biochmn. L?iojiIi~~,s. Re%. Conmii~r. 50,

6. Wilson. M . 7.. (;reenwood. C., Brunori. M. & Anlonini. t:. (1975)

Ci. & Mondovi, B. (1970) J . Bid . Chcr??. 24.5. 4847-4849.

8 5 3 - 8 5 8 .

Biochriir. J . 14.5, 449-457,

344

[Cyt(III)]-' + [Cyt*(III)]-' - [Cyt(II)]-'

[Cyt(III)]- [Cyt(II)]-' + [Az(II)]-' + [Cyt(III)]-' + [Az(I)]-'

Further according to Castellan: T;& = rslOw g2/g, = ralnw (g22 . g,, - g:J/gll.

T,:, = k12 . [Cyt(II)] [Az(II)] . {[Cyt(II)]-' + [Az(II)]-' + [Cyt(III)]-' + [Az(I)]-'} { [Cyt(III)]-' + [Cyt*(III)]-'} - j[Cyt(III)]-' ' [Cyt*(III)]-l)/{ [Cyt(III)]- + [Cyt*(III)]- 1 1

= k12 . {[Cyt(II)]-' + [Az(II)]-' + [Cyt(III)]-' + [Az(I)]-') {[Cyt(II)]-' . [Az(II)]-'} - k12 . {[Cyt(III)]-' . [Cyt*(III)]-'}/{Cyt(III)]~' + [Cyt*(III)]-'] . [Cyt(II)] . [Az(II)]

= k12 . {[Az(II) + [Cyt(II)] + [Cyt(II)]/[Cyt(III)] . [Az(II)I + [Az(II)]/[Az(I)] . [Cyt(II)]] - ki2 . ([Cyt(II)] . [Az(II)]J/{ [Cyt(III)]

= k12 ([Az(II)I + [Cyt(II)] + [Az(I)] [Cyt(II)] . [Az(II)]/[Cyt(III)] . [Az(I)] + [Cyt(lII)] [Az(II)] . [Cyt(II)]/[Az(I)] [Cyt(III)])

[Cyt*(III)] .([Cyt(III)]-' + [Cyt*(III)]-')}

- k12

= k12 . {[Az(II)I + [Cyt(II)I} + k12iK2 {[AZ(~)] + [Cyt(III)I} -k12 . [Az(I)]/K~ (1 + K o ' )

= k12

= k12 . ([Az(II)I + [Cyt(Ii)l) + k21 { [Cyt(III)] + [Az(I)] . [I - l / ( l + KC')]}

= k12 . {[Az(II)l + [Cyt(II)]) + kzl ([Cyt(III)] + [Az(I)]/(I + KO)}

{[Cyt(II)I . [ W W I . [ M I ) l } / { [Cyt(IWI . [Az(I)I .([Cyt(II)I/[Cyt(III)I + [C~t(III)l/[Cyt*(Ili)1):

{[Az(II)] + [Cyt(II)]] + kzl ([Az(I)] + [Cyt(III)]} - k21 [Az(I)]/(1 + KC')

which is identical with Eqn (3)

7. Rosen, P. & Pecht, I. (1976) Biochemistry, 15, 775-786. 8. Wherland, S. & Pecht, I. (1978) Biochemi.yrry, 17, 2585-2591. 9. Goldberg, M. & Pecht, I. (1976) Biochemistry, 15, 4197-4208.

10. Dickerson, R. E., Timkovitch, R. & Almassy, R. J . (1976) J . Mol.

11. Takano, T. & Dickerson, R. E. (1980) Proc. Naif Acad. Sci. (',Y I .

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P. Rosen, M. Segal, and I . Pecht, Department of Chemical Immunology, The Weizmann Institute of Science, P.O. Box 26, IL-76-100 Rehovot, israel