Photodissociation of dimethylnitrosamine

INTRAPROTEIC

1 May 1981

CXEMICAL PHYSICS LETTERS

Volume 79, number 3

LIGAND DIFFUSION

IN LASERPHOTODI!3SOCIATED

CARBOXYHEMOCLOBIN

AND SUBUNITS

L. LKNDQVIST, S. EL MOHSNI *, F. TFIBEL bboratoue de Photophysque Moi&zA-aue du CNRS *. Umwrsire PartMud. 91405 Ormy Cedex. Fknce

B. ALPERT hborarolre

de Biologre Phynco-Chrmlque.

UER Blomtfdicale des Samt P&es, 75270 Pans Cedex 06, fiance

and J-C. ANDRE Laboratoue de &imre G&&ale, ERA No. I36 du CNRS, 54042

Nancy

Cedex, France

Received 8 !anuary 1981

Carboxyhemoglobin in aqueous solution was photolssoclatcd by laser pulses of l-7 ns at 530 nm. KmeQc spectrophotom. etry showed that gemmate recombmatzon of CO to the heme, occurrmg durmg the fust 100-200 ns after ~IS.SOCMI~II, has the square-root-of-tune dependence expected for hgation durmg random-walk diffusion of the &and m the protern. Isolated Q and P subumts gave similar resuits.

l_ Introduction

m the nanosecond tune range followmg elation of carboxyhemoglobin (HbCO)

It IS weLl known that the oxygen-complexmg efficiency of the heme in respiratory hemoproteins IS strongly dependent upon the protelc part of the bmmolecule. Hindrance of ligand access to the heme due to the protein structure is considered as one of the factors contnbuting to this dependence [ I]. However, expenmental eviderce for such a shielding effect is scarce. Frauenfelder and co-workers [2,3] studled

the rebincih?g kinetics after photodlssociatlon of Eganded hemoproteins in glycerol-water mixtures, particularly at low temperatures. They concluded that ligand displacements in the heme pocket are govarned by a number of energy barriers, corresponding to d& ferent regions or sites of this cavity. Alpert et al. [4] reported the existence of geminate pair recombination

* Also at. Laboratolre de Biologie PhyslcoKhirnique, UER l

photodlsso-

in aqueous solution at room temperature, and they proposed that this reaction occurs durmg random diffusion of the hgand through the protem. Sunrlar results were also found for the isolated OLand p chains [S] . These fiidmgs directly demonstrate the ligand imprisoning effect of the proteic mattix. Geminate recombmation of phototissoclated oxyhemoglobin was observed recently by Chernoff et al. [6] in the 200 ps range. These authors discuss the possibtity of single-site trappmg of the &and versus random-walk

diffusion

in the protein.

However,

the

kinetic data did not make possible a chstmctton between these two mechanisms_ We report III the present paper a kinetic study which shows that the nanosecond recombmation [4,5] has the time dependence expected for religation governed by random dlffusron inside the protein molecule.

Biom&Iicale des Saint P&es, 75270 Paris Cedex 06. France. Laboratory asoclated with Universtd Paris-Sud.

0 009-2614/8

1 /0000-0000/$02.50

Q North-Holland

Publishing Company

525

Volume

79. number

3

CHEMICAL

PHYSICS

2. Experimental

Hb and Its Isolated subunits were prepared from fresh human adult blood [7,8 1. Aqueous solutions of the proteins at pfT= 7.U (0.1 M potassium phosphate 6utXx) were degassed by pumpmg, and a mcnute amount of potassium &thromte was added to remove the last trsces of Oll~eTr. The CO comp&Les were obtamed by admission of CO above the solutions to atmosphenc pressure. The second harmomc (530 nm) of the emlsslon from a Nd/glass laser was used for photodlssocration A Pockels cell isolated laser pulses of 7 ns width at halfmaximum. The opemng and closmg time of the cell was l-2 ns, g~vmg a total pulse width of = 10 ns. Pulses of 1 5 ns fwhm were also used III a few runs The laser energy mcldent m the sample was ~30 mJ. Complete photodlssoclation of the protein-CO complexes was obtained at this energy at the concentrations used The rate of recombmatlon was measured by kmetlc spectrophotometry kvlth a tune resolution of 1-2 ns as described previously [4]. A double-beam oscdloscope was used to record simultaneously absorbance changes over a trme penod of 0.2 and 1 ps, respectively, after dlssociatlon. The data reported represent the mean of 6 runs per protem.

3. Results

and discussion

Absorbance changes due to photodlssociatron of the CO-protem complexes were measured as a function of time after laser pulse excltatlon at the wavelength of the Soret peak. The absorbance (A0 before rrradlatlon) decreased at the onset of the laser pulse (t = 0), reaching a muurnum value (A,) approxunately at the end of the pulse (t = te) due to formation of the less-absorbing free protem. Durmg the foliowmg 100-200 ns the absorbance Increased agam, attainmg a value (A,) mtermediate between A,-, and A,. The absorbance remamed at this value for several mlcroseconds. We prevrously showed [4] that the raped change (also observed by Duddell et al. [9,10] ) corresponds to recombmatron of photodehgated CO mth gemmate heme and proposed that this refixatron occurs durmg diffusion of the hgand in the protem molecule. A fractlon T_ = (A, - A,)/(A, - Ao) of the dtssoclated hgand recombtnes because of tlus reactton, the remamder es526

LETTERS

1 May

1981

capmg from the pratem Into the aqueous phase. We now have fbund that the kmetlcs of recombmatlon for Hb and the Isolated suburns can be expressed by the relatton

r>

te_

(1)

The stratght lmes obtarned on plottmg log(A_ - A) against cl/-?, as shown m fig. I, demonstrate the validtty of this expression Values for I-, and the rate parameter a are given m table 1 Eq. (1) was found to hold between 4 and 38°C The choice of time orlgm IS somewhat arbitrary It was found that the use of the laser pulse departure as orlgm gave the best ilt of the experlmental curves to expresston (1) Thus 1s probably because most of the complex IS dlssoclated already durmg the pulse rise time at the strongly saturatmg mtensltles used rn the present study. In a few runs, the lmtlal phase of the recombmatlon was studled for Hb also usmg laser pulses of 1 5 nm fwhm. The result, shown m fig I, shows that

-‘-

7

:

a

Y -15-

-2 -

FIN 1. Absorbance changes observed on photodlssoclauon of CO from Hb (o), subumt a (A), and subunit p (o), plotted agamst the square root of tune after the onset of the laser pulse (The curve for the p subumt IS dlsplaccd upwards by 0 5 umts.) Temperature 24OC. heme concentration =40 PM, optlcal path I mm, laser pulse width 7 ns fwhm, momtormg wavelength 419, 419 and 420 run. respectwely. The absorbance change for HbCO at a laser pulse width of 1 5 ns fwhm LSalso shown (- . - . -)_

CHEMICAL PHYSICS LiXl-ERS

Volume 79. number 3

Table 1 Parameters determmmg the gemmate recombmauon of the photodlssoclated CO-hemoprotems at 24% Estunated accuracy ofa

2.58, of rm- tO.02

Protem

II (lo7 s-l)

rrn

Hb L1

55 6.4 39

042 0.29 0 27

P

the ttl2 dependence is vahd at least down to 3 ns after the dlssoaatlon. It may be mentioned that Chernoff et al [6] recently reported results of a laser study of HbCO at picosecond resolution These authors found the recombmatlon to be neghglble durmg the first I.2 ns after the dlssoclatlon From the slopes of the curves m fig. I one would mdeed expect to obtain at the most 10% recombination (limit of detection of the plcosecond study) during this time interval The observed t112 dependence suggests that the gemmate recombmatlon IS determined by the probabdreencounters occurring durmg rty of hgand-Fe2+ random dlffuslon of the photodehgated hgand through the protein. However, complete randomness cannot be expected smce the protein mterlor IS heterogeneous (although rapid structural fluctuations [I I- 131 must attenuate the effect of this heterogeneity to some extent). We have performed theoretlcal calculations [ 141 of the recombination kinetics expected for one- and three-dlmenslonal diffusion of the hgand to determine the effect of dIffusIona amsotropy on the recombmatlon. It was found that the hnetrcs obtained wrth any of these models can be fit satrsfactordy to the analytrcal function (I ) durmg the maJor part of the recombmatron It follows from the above results that the geminate recombmatlon can be dlvlded mto two processes. (1) mtraprotelc hgand diffusion, and (2) reaction durmg encounters of ligand with geminate Fe2+. The parameters Q and r, depend upon the rates of both these processes. It IS seen from table 1 that the r_ values for the two subunits are closely the same whereas the Q values are different Pius resuIt indicates that both the &ffusion rates and the rates of reaction during gemmate pair encounters are different for these proteins. The a value for Hb IS approxuTlately the mean of those for the isolated subumts; however, r, IS higher. Thus dlfference shows that both the hgand diffusion rate and

1 May 1981

the heme reactlvlty are affected by the assoclatlon of the subunits to form Hb However, a more detalled analysis IS required to determine these effects quantatatively The ttl2 dependence characteristic of gemmate recombmatlon kmetrcs was previously observed by Hasmoff [ I5 J m a study of the photodIssociation of myoglobm-CO m glycerol-water glass at -78°C by flashes of 0 I-O 7 ms duration This author concluded that a cage effect m thus case IS produced by the solvent because of Its extremely high vlscoslty Frauenfelder and co-workers, on the other hand, obtained multiexponential recombmatlon kmetlcs for myoglobm [I?] and for the subunits of Hb [3] m condltlons similar to those of Hasmoff They attributed these kinetics to the existence of a number of trapping regions or sites III the heme pocket These authors also studied the recombmatlon at room temperature However, the duration of the photolysls pulses was too long (1 11s) to allow observation of the geminate reaction at this temperature, and mono-exponential recombmatlon (due to CO dlffuslon from the aqueous phase) was therefore obtamed We show m the present study that multiple hgand trapping is not important at room temperature The observed non-exponentrahty arises under these con&tions because the average distance between the reactants Increases gradually durmg essentially random diffusion of the hgand m the protein. This varlatlon m spatial dlstrlbutlon of the hgand around the reactive site probably has to be considered also m the analysis of the lowtemperature kinetics

References

[ 11 hl F Pcrutz. Nature 228 (1970) 726. [21 R H Austm. K W Beeson, L Elscnstcm, H. Frauenfelder and I C Gunsalus. Biochemistry 14 (1975) 5355 [3] N Alberdmg. S S Chan. L. Elsenstem. H rrauenfelder, D Good. 1 C Gunsalus, T hl Nordlund, hl F Perutz, A H Reynolds and L B Sorenson, Blochemlstry 17 (1978) 43 (41 B Alpcrt. S El klohsni. L. Lmdqwst and F Tfibel, Chem Phys Letters 64 (1979) 11 I.51 B Alpert, L. Lmdqwst. S. El hlohsnl and F Tfibel, m Interactions between uon and protems m oxygen and electron transport, ed. C Ho (Elsevrer. Amsterdam), to be pubhshed. [6l D A. Chernoff. RM. Hochstrasser and A W. Steele, Proc. Natl Acad. SCI. US 77 (1980) 5606.

527

I’trme 79, o_umkt

,,7] ‘fgj ! [9]

C

cHEMSCALPHYS~CSI;ETTERS

M.F. Perutt, 3. Crystal Grow& 2 (1968) 54. G. Geraci, LJ. Parkhurst and Q.H. Gibson, J. Bial. Chem. 244 (1969) 4664. D.A. Duddell, R J. Morris and J.T. Richards, 3. Chem. Sot. Chem. Commun. (1979) 75. 101 D.A- Duddell, RJ. Morris and J-T. Richards, Biochem. Biophys. Acta 621 (1980) l-

1 May lP81

[ill f. Lakowia and G. Weber, Biochemistry 12 (1973) 4171. [l?!l 3.A. McCammon, B-R. Gelin and M. Karplus, Nature 267 (1977) 5612. f13] W. Frauenfelder, G.A. Petsko and D. Tsernoglou, Nature 280 (1979) 558. 1141 L. Lmdqvist, S. El Mohsni, F. Tfibel, B. Alpert and J.C. Andre, to be published.