Erythrocruorin: a Possible Model (Proteins/Steady State/Photochemical Efficiency) G
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Proc. Nat. Acad. Sci. USA Vol. 72, No. 11, pp. 4313-4316, November 1975 Biochemistry Kinetics of binding of carbon monoxide to Lumbricus erythrocruorin: A possible model (proteins/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 protein 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 hemes 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 (heme); (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.