Proc. Nat. Acad. Sci. USA Vol. 72, No. 11, pp. 4313-4316, November 1975 Biochemistry

Kinetics of binding of carbon monoxide to Lumbricus : A possible model (/steady state/photochemical efficiency) G. M. GIACOMETTI, A. FOCESI*, B. GIARDINA, M. BRUNORI, AND J. WYMAN C.N.R. Center of Molecular Biology, Institutes of Chemistry and Biochemistry, Faculty of Medicine of the University of Rome and the Regina Elena Institute for Cancer Research, Rome, Italy Contributed by J. Wynan, August 18,1975 ABSTRACT This paper represents a kinetic study of the A millimolar stock solution of carbon monoxide was pre- binding of carbon monoxide by Lumbricus erythrocruorin. pared by equilibrating degassed distilled water with gaseous Observations on the quantum yield and the relaxation of the CO at 1 atm (101.3 kPa) of partial pressure at 200. system both to equilibrium and to the steady state realized in The intensity of the photodissociating light, both in the the presence of constant illumination under various condi- flash and in the steady-state (continuous light) experiments, tions are reported. The results, besides indicating the exis- filters. The photolysis tence of at east two types of binding sites, give indications was controlled by the use of neutral as to the behavior of a complex polyfunctional molecule, apparatus was the same as that previously described (4). such as an enzyme, working under steady-state conditions. Kinetic experiments were analyzed with a Hewlett-Pack- ard model 9830 A desk computer. Photochemical efficiency This paper is a sequel to an earlier one on the binding of car- was determined by the "pulse method" previously described bon monoxide by erythrocruorin, the giant respiratory pro- (5). tein of Lumbricus, under steady-state conditions produced by a photodissociating light (1). Both papers are relevant to RESULTS an understanding of the mode of functioning of an enzyme, 1. Flash photolysis experiments for all enzymes operate under steady-state rather than equi- The relaxation after an intense flash, which completely dis- librium conditions, and together the two papers provide a sociates the ligand, was studied under two sets of experimen- touchstone for judging certain of the ideas put forward in a tal conditions: recent communication in these PROCEEDINGS (2). of the was com- es- (a) When the initial saturation The results of the earlier paper (1), which represent an plete, Y = 1. In these experiments the range of total concen- sentially static approach to the problem, may be summa- tration of CO extended from 20 IMM to 100 MM, and the con- rized as follows: centration of free CO can be regarded as having been con- (i) The spectral measurements show the absence of any stant throughout the relaxation process, which was approxi- exact isosbestic points in the binding of CO to deoxyerythro- mately first-order in CO concentration. cruorin such as would be expected in the case of a simple (b) When the initial saturation of the protein was low, Y process. 0.2. Here the concentration of free CO decreases during (ii) The shape of the binding curve (or Hill plot) is pro- relaxation, which now appeared as pseudo first order in sites foundly modified under the steady-state conditions pro- concentration. duced by the stationary light, the cooperativity being re- In case a the relaxation was found to be strongly biphasic, duced. being partly fast and partly slow. In case b it was monophas- (Mi) The quantum yield, as determined by the "pulse ic, being all fast and similar to the fast phase observed in method," varies, though not greatly, with the saturation of case a. Both sets of experiments were analyzed in term of a the molecule with CO, passing through a maximum in the second-order rate constant 1'. The results, in terms of 1' as a middle range of saturations. All these observations were function of the percent reaction, are shown in Fig. 1. From shown to be phenomenologically consistent. a the reaction to an analysis of the data it appears that in case The present study, which represents a kinetic approach was about 70% slow, with 1' = (1.9 4 0.6) X 105 M-1 sec-1, the same problem, describes observations on the quantum and 30% fast with 1' = (1.4 I 0.4) X 106. In case b, where yield and the relaxation of the system both to the steady the process was all fast, 1' = 1.5 X 106 M-1 sec-1, the same state and to equilibrium under various conditions. as in case a. MATERIALS AND METHODS 2. Action spectra Earthworm erythrocruorin was prepared as previously de- Action spectra for the fast and slow phases of the reaction scribed (3) and kept at 4°. All experiments were made in 0.2 calculated from the oscillograph traces under conditions a M phosphate buffer at pH 7.4, where the protein is stable in are shown in Fig. 2 and are consistent with the lack of a true its highest degree of aggregation (molecular weight 3.3 X isosbestic point reported earlier (1). From Fig. 2 it is clear 106, corresponding to about 140 per molecule). For that the determination of the relative amounts of fast and each preparation the state of aggregation of the protein was slow components must depend on the observation wave- checked by ultracentrifuge analysis. Protein concentrations length. The figures, 70 and 30%, given above were based on were determined spectrophotometrically using EmM = 113.6 measurements far removed from the point of crossing of the at X = 430 nm for the deoxygenated derivative (3). curves in Fig. 2. It was found that if the intensity of the flash was gradually decreased so that only partial dissociation was * Fellow of Fundacao de Ampero a Pesquisa de Estado de S. Paulo achieved, the results, both as to the relative amounts of the (Brasil). Proc. quimica 73/1178. two components and their rate constants, were unaffected. 4313 Downloaded by guest on September 29, 2021 4314 Biochemistry: Giacometti et al. Proc. Nat. Acad. Sci. USA 72 (1975)

A t(B) AAAA 0.5k 10 F A 0

.- *n3 4) x) 2 -0 wA 0

(A) 0

o L 0.5 0 20 40 60 80 100 410 420 430 440 % REACTION (nm) FIG. 1. Dependence of the apparent second-order rate constant 1' for CO combination (ordinates) on the percent reaction V (ab- FIG. 2. Kinetic difference spectra for the fast and slow compo- scissas) as obtained from flash photolysis experiments on CO- nents in the combination of CO with erythrocruorin. Insert shows erythrocruorin starting from: (A) fully saturated erythrocruorin, the oscilloscope trace of a flash photolysis experiment performed CO concentration = 25 mM and protein concentration = 2.8 mM at X = 426.5 nm (see arrow), where the two reactions have opposite (); (B) partially saturated erythrocruorin (Y = 0.2), CO con- absorbance changes. Other conditions as in legend of Fig. 1. centration = 0.3 mM and protein concentration = 1.45 mM (heme). Conditions: pH 7.4, 0.2 M phosphate buffer, and about 200. X = 436 nm. (A Y _ 0.2). If the photodissociation is achieved with a brief pulse of light (duration n 100 ,usec), the time course of CO combination in the dark is biphasic, with a ratio of fast to 3. Relaxation kinetics: light-to-dark transition slow sites equal to 30/70 at X = 436 nm (as given in Section A continuous light incident on the protein solution fully sat- 1). If illumination is maintained for a longer time (more urated with carbon monoxide generates a steady state with Y than 1 sec), the recombination in the dark is monophasic and < 1. (1, 6). The relaxation from the steady state in the light corresponds to the slow kinetic component only. When the to equilibrium in the dark when the light was suddenly pulse of light is continued for a finite, intermediate time (in turned off was carefully examined. Under all conditions ex- Fig. 4 it lasted 60 msec) and a steady state is not reached, the plored, the approach to equilibrium was found to corre- recombination in the dark still shows a biphasic behavior, as spond to a simple relaxation process, with a second-order evident from Fig. 4. combination rate constant identical to that observed for the slow phase as reported above (see Fig. 3). This finding indi- 4. Relaxation kinetics: dark-to-light transition cates that the faster sites, which are preferentially populated The opposite relaxation, i.e., the approach to the steady state at low saturation, are the ones that are largely ligand-bound in the light starting from the dark, was also analyzed. Under under steady-state conditions at fairly high saturations, and therefore are not observed. If this is the case, one should expect an enhancement of the relative amount of the fast component by flashing off the ligand still bound under the steady illumination. Such expectation was confirmed by the experiment summarized in Table 1. This also revealed a 2-fold increase in the rate constant of the slow process, which was reduced in ampli- tude, but still present under these conditions (Table 1). Fig. 4 reports the results of an experiment that confirms 0) the findings given above. A solution of fully saturated a, erythrocruorin (Y = 1.0) was subjected to a pulse of light sufficient to photodissociate only a fraction of the total sites

Table 1. Enhancement of amount of fast component under stationary illumination* l'(slow) l'(fast) 10-5 10-5 0 x x 0 20 40 60 80 (M-1 sec-') (M-' sec1-) % Slow [CO + HEME] M Flash from the dark 1.9 12 69 FIG. 3. Dependence of the first-order rate constant for CO Flash from the combination as a function of the CO and protein concentrations. stationary light 3.0 15 58 (A) Results obtained by flashing a solution of erythrocruorin par- tially saturated with the ligand (Y = 0.06); (B) results obtained in * The reported values are the average over different flash intensi- the light-to-dark transition starting from a fully saturated solution ties ranging from 1.0 to 0.06. of erythrocruorin. Other conditions as in legend of Fig. 1. Downloaded by guest on September 29, 2021 Biochemistry: Giacometti et al. Proc. Nat. Acad. Sci. USA 72 (1975) 4315

,. 0 -I-I

ITESfITY FIG. 5. Dependence of the first-order rate constant for the ap- A . proach to the steady state (dark-to-light transition) as a function 20 40 60 80 100 120 of relative light intensity. Numbers given in the figure indicate the TIME (msec) final saturation values reached at the various light intensities. FIG. 4. Time course of recombination of erythrocruorin with CO after light pulses of different duration. (A) Flash of 100-Msec high affinity one-just the opposite of what would be ex- duration, (dark) = 1, A R = 0.41; (B) stationary light pulse of pected. As a reasonable explanation of an otherwise puzzling Y = A Y = 0.12; stationary light 60-msec duration, (dark) 1, (C) situation, we suggest the following simple model: the 140 pulse of 1-sec duration, R (dark) = 1, AY = 0.34. Protein concen- are into tration = 2.8 MAM (heme), and CO concentration 25 MM. Other con- odd sites (hemes) of this huge molecule segregated ditions as in legend of Fig. 1. constellations, essentially independent of one another, each being the seat of strong cooperative interactions resulting the conditions explored a monophasic relaxation was always from local ligand-linked conformational changes [a possibili- observed; the reciprocal relaxation time increases with light ty suggested in a recent paper (8)]. These constellations are intensity, as shown in Fig. 5. of two types, one consisting of fast high affinity sites, the It should be remarked at this point that there is good evi- other of slow low affinity sites, the median ligand activity of dence that the dissociation rate constants for CO are very the former being greater than that of the lattert. Though the small. Thus, a measure of the dissociation rate constant for constellations are independent, there is a considerable over- the fully saturated molecule has been obtained from re- lap in their binding ranges, and the observed cooperativity placement of CO by oxidation with ferricyanide. The value of the whole system is the resultant of the very strong coop- obtained was found to be 1 = 3 X 10-3 sec1. In addition, erativity of each constellation and the opposing effect of the the data given in Fig. 3 (experiments performed at very low heterogeneity of the constellations. In accordance with this saturations) yield an upper estimate of 1 for the higher affin- model the observed relaxation time will, under all condi- ity sites; this value cannot be higher than about 1 sect and tions, be a combination of the relaxation times of the two is probably well below. types of constellation, which in each case we assume to be 5. Photochemical efficiency characterized by a single predominant eigenvalue resulting from the solution of the secular equation-fast in the case of The photochemical efficiency was measured by the "pulse the high affinity constellations, slow in the case of the low method" (5) for solutions fully saturated with CO, both affinity onest. This picture is reasonably consistent with all under equilibrium conditions (in the dark) and under the facts, which may be summarized as follows: steady-state conditions characterized by an average photo- (i) The lack of any true isosbestic point in both static and dissociation of about 30% (continuous flux of light). No sig- action spectra is clearly established. This would be expected nificant difference was observed between the two condi- from the number of different forms, of which there would tions. It is worth remarking that in the light the photochemi- be at least two for each type of constellation. cal efficiency for the fast species should be preferentially (ii) Recombination of the ligand after a flash applied to measured, whereas in the dark an average between fast and the system at equilibrium is biphasic, the proportion of fast slow sites is observed. The coincidence of the two values is phase increasing with decreasing Y (at full saturation, when however not inconsistent with the values of quantum yield Y = 1, about 70% of the system is slow; when Y is less than at low and high saturations, as determined in the previous about 0.2, the system is nearly all fast). This accords with the paper (1). idea that the high affinity constellations are the fast ones. The fact that the ratio of fast to slow fractions observed DISCUSSION t More than two types of constellation could of course be present, It may that the cooperativity of ligand be safely assumed but to explain the data it suffices to consider only two. binding (nmax _ 4) shown by the giant erythrocruorin mole- fIt should be pointed out that when Y = 1 and free CO is present cule represents an allosteric effect. If, therefore, erythrocru- in large excess, the equations developed in the preceding paper orin constituted a simple two-state system (7), which it al- (2) are strictly applicable to each constellation. On the other hand, most certainly does not, we should expect that the low affin- when Y < 1 and free CO is present at lower concentration, the li- ity form would predominate at low saturations and be grad- gand concentration will vary during the relaxation process and the underlying assumption that the X's in Eq. [1] of that paper are ually replaced by the high affinity one as liganding proceed- constant no longer holds, i.e., the equations are no longer linear ed. Actually, although measurements of the off constants are and the exact form of their solution cannot be given. Neverthe- difficult, it looks as if 1 for the fast form were either greater less, the experiments show that relaxation to a steady state still oc- than, or at least similar to, that for the slow one. If so, then curs, as would be expected, and it seems reasonable to analyze the the fast form, predominant at low saturations, would be the results in terms of the same set of concepts. Downloaded by guest on September 29, 2021 4316 Biochemistry: Giacometti et al. Proc. Nat. Acad. Sci. USA 72 (1975) under these conditions is insensitive to the intensity of the ure) does in fact correspond to the previous observation that flash (i.e., to the amount of ligand driven off) would imply the quantum yield for CO depends on the initial saturation, that there is no great difference in quantum yield between passing through a maximum in the middle range. Unfortu- the sites in the fast and slow constellations. This is in accord nately, apart from the complexity of the situation, the pic- with the relatively slight variation in overall quantum yield ture here is somewhat marred by the dependence of 1' (for at the lower and upper limits of Y previously reported (1). the slow form at least) on the intensity of the steady light. (ii) When a flash is superimposed on steady illumination, A significant point in relation to the recent paper (2) is the the subsequent recombination process is enriched in fast interpretation of the maximum in the curve of quantum component. In this case, the quantum yield of the fast and yield versus saturation. Assuming that there are only two slow forms being not very different, the ratio of fast high af- kinds of site (constellations), this would demand a difference finity to slow low affinity constellations will increase in the of the quantum yield of a given type of site (fast or slow) de- steady state created by the light, provided, as we may sup- pending on its conformation (saturation). The high affinity pose, that (1) fast > (1) slow. sites, which are liganded first, would have to have a higher (iv) The relaxation when a stationary light is suddenly quantum yield in the liganded than in the unliganded con- turned off is slow. This would follow from the fact that in formation. For the low affinity sites the situation would be the presence of the light it is mainly the slow constellations the opposite§. This dependence of quantum yield on confor- that are unliganded. Here the effect of the light is the con- mation is precisely the condition for the "circulation" under verse of that in the last paragraph. steady-state conditions discussed in the preceding paper, as (v) When the light is suddenly turned on, the relaxation to the "turning wheel" phenomenon. It would certainly be of the steady state shows a single relaxation time, which in- interest in this connection, if it were possible, to see what creases with light intensity. This means that in the presence happens to the heterotropic linkages (e.g., the Bohr effect) of the light the relaxation times of the two constellations be- under steady-state conditions. Unfortunately, at pH values come approximately equal. This is what would in a general much removed from neutrality the protein undergoes irre- way be expected. Thus, in the simple case of , it is versible transitions (9). Of course the erythrocruorin mole- confirmed that cule is rather different from the hypothetical working en- zyme considered in the earlier paper, but it is not perhaps IT = l+wI+l'(CO+Fe++) [1] too different to provide a prototype of what might be ex- pected. I w where is light intensity and represents photochemical ef- 1. Giacometti, G. M., Focesi, A., Brunori, M. & Wyman, J. (1975) ficiency. In strong light, therefore, 1/i approaches W I. In J. Mol. Biol., in press. erythrocruorin the data show that w is similar for both con- 2. Wyman, J. (1975) Proc. Nat. Acad. Sci. USA 72,3983-3987. stellations, so that in strong light the relaxation time of both 3. Rossi Fanelli, M. R., Chiancone, E., Vecchini, P. & Antonini, E. constellations should become approximately the same. Of (1970) Arch. Blochem. Blophys. 141, 278-283. course the situation is not nearly as simple as in myoglobin 4. Bonaventura, C., Bonaventura, J., Antonini, E., Brunori, M. & since these are two different kinds of constellation, each of Wyman, J. (1973) Biochemistry 12,3424-3428. which undergoes an allosteric transition. (This is represented 5. Brunori, M., Giacometti, G. M., Antonini, E. & Wyman, J. in the fact that 1/r is not, as in the case of myoglobin, strict- (1973) Proc. Nat. Acad. Sci. USA 70,3141-3144. ly linear in I.) But the interpretation would seem to be es- 6. Brunori, M., Bonaventura, J., Bonaventura, C., Antonini, E. & Wyman, J. (1972) Proc. Nat. Acad. Sci. USA 69,868-871. sentially correct. 7. Monod, J., Wyman, J. & Changeux, J. P. (1965) J. Mol. Biol. (vi) The value of 1' for the slow form increases by a factor 12,88-109. of roughly 2 in the presence of steady light. Dependence of 8. Colosimo, A., Brunori, M. & Wyman, J. (1974) Biophys. Chem. 1' on the presence of light would be predicted in accordance 2,338-344. with our model since the ratio of the allosteric forms of each 9. Giardina, B., Chiancone, E. & Antonini, E. (1975) J. Mol. Biol. constellation is different in the steady state and at equilibri- 93, 1-10. um. (Data on the effect of light on 1' fast are lacking.) Assuming that Eq. [1], which holds for the simple case of § It must be admitted that the same dependence of quantum yield myoglobin, applies also to erythrocruorin, we should expect on saturation could also be explained by postulating three or more the slope of the curve in Fig. 5 to give, at any point, the pho- types of site (constellations) differing from one another as to tochemical efficiency w. It is suggestive that the change of quantum yield without assuming a dependence of quantum yield slope on the final saturation with CO (also given in the fig- on conformation in a given type of site. Downloaded by guest on September 29, 2021