PHYSICS OF BIOMOLECULES

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Submitted on 1 Jan 1974

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PHYSICS OF BIOMOLECULES

H. Frauenfelder

To cite this version:

H. Frauenfelder. PHYSICS OF BIOMOLECULES. Journal de Physique Colloques, 1974, 35 (C1),

pp.C1-65-C1-66. �10.1051/jphyscol:1974121�. �jpa-00215496�

JOURNAL DE PHYSIQUE

Colloque

C1,

supplkment ail

¹¹⁰

1, Tonie 35, Janoier 1974, page

C1-65

PHYSICS OF BIOMOLECULES ^(*)

H. F R A U E N F E L D E R

University of Illinois, Urbana, Illinois, USA

R6sumC. -

La structure des bion~olecules est tracke et la reaction des proteines heme avec du monoxyde de carbone est discutee. La myoglobine ferreuse, par exemple, se lie avec CO. Le CO peut btre retire par un flash de luniiere visible et on peut alors etudier la combinaison. Celle-ci s'effectue jusqu'g une Tde 50 K. En dessous de 150

K,

elle n'est pas exponentielle. La dependance de temps observee,

H ( t ) = H ( 0 ) (1 -1- t / t , ) J f ,

s'explique en supposant que I'energie d'activation est decrite par un spectre d'knergie,

g ( E ) .

Abstract.

-

The structure of biomolecules is sketched and the reaction of heme proteins with carbon monoxyde is discussed. Ferro~is niyoglobin, for instance, binds CO. The CO can be removed by a flash of visible light, and rebinding

can

then be studied. Rebinding occurs down to a tempera- ture of 50 K. Below 150 K,

it

is not exponential. The observed time dependence,

is explained by assuming that the activation energy

E is

not sharp, but is described by

an

energy spectrum,

g(E).

Biomolecules form a part of physics

;

their structure and their reactions can be studied with physical tools and can be described in physical terms. Proteins, in particular, are crucial for an understanding of biolo- gical phenomena in physical terms. In this lecture, I will sketch the general structure of proteins [I] and describe recent low-temperature studies of biomole- cular reactions.

A comparison between solids and biomolecules shows some fundamental differences. Solids are truly three-dimensional systems. Each atom in a solid is linked more-or-less i~niformly in all three directions.

Translational syni~iietry simplifies tlie theoretical understanding. Proteins, in contrast, are essentially linear structures. The building blocks of proteins, 20 different amino acids. are linked together by peptide bonds to form a long linear chain. This PI-imary structure is fully characterized by tlie sequence of amino acids.

In the proper solvent. s ~ ~ c l i a linear polypeptide chain folds to form a three-dimensional structure. In the proteins of interest here, this s t r u c t ~ ~ r e is globular, with linear dimensions of the order of 50 A. The struc- ture is held together by hydrogen bonds and disulfide bridges between various parts of the chain. Two features of the globular protein are crucial, tlie pocket and the active center. Tlie pocket is tailor-made to accept and recognize tlie lnolecule on which the pro-

(*) The

work reported

hcrc was

pet.formcd

in

collaboration

with R. H.

Austin,

K.

Bceson,

L.

Eiscnstcin,

1. C.

Gunsalus

a n d V. P.

Marshall.

It was

supportcrl

in part by

Grants

fl-om the

National Science Foundation

NSF

GP

26281 and I-lt'U C;b1 18051

and AM

00562.

tein works, and the active center some\vhe~-e in the pocket is where tlie work is performed.

In the following, I will only be concerned with one class of biomolecules. namely the heme proteins.

Probably tlie best known example of this class is hemoglobin, the oxygen carrier in the blood [2].

Myoglobin (Mb), which stores oxygen in the musclcs.

is another example. Many important enzymes (cata- lysts) also belong to the same group.

All heme proteins have basically the same acti\e center, namely

hen?(.

(ferrous protoporphyrin). In Iienie, an iron atoni is bound to f o ~ ~ r nitrogen ~ttonis

~ ~ h i c l i in turn belong to four pyrrole rings. Because of the iron. heme proteins have been a favorite hunting ground for Mijssbauer physicists. Since this subject will be treated in another lecture, I will not discuss

it

here.

Many heme proteins in the ferrous state. particularly those that bind oxygen, form stable complexes with carbon monoxide (CO). The C O binds as a n axial ligand to the iron. Tlie linkage can be broken by irra- diation with visible light. C O then rebinds again. Thc photodissociation atid the subsequent rebinding can be observed optically since the absorption spectra oS the CO-bound and the free ferrous heme proteins are dif- ferent. The kinetics of the rebinding has been observed in many experiments. However, nearly all of these experiments liavc been performed in a

rial-row

tcmpe- ri~tul-e range betlveen about 770 K and -300 K . We have

~.ccently extendeci me:tsilrements down to r n ~ ~ c l i IO\VCI- temperatures by. using heme

~ I - O ~ C ~ I I S

in glycerol-

\\.:~ter solutions [3]. These expcrin~cnts reveal

L I I ~ S L I ~ -

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1974121

C1-66 H. FRAUENFELDER

pected phenomena and the essential features will be described here briefly.

Rebinding of CO to Mb after removal by a light flash occurs down to 50 K. Four regions of different kinetic behavior occur. Above 240 K tlie reaction is second-order

;

between 240 and 200 K rebinding becomes exponential and independent of CO concen- tration. N o signal is seen between 200 K and 150 K.

Below

150

K, the reaction can be observed over many orders of magnitude in time and it is described appro- ximately by

where H ( t ) is the normalized number of M b mole- cules that have not rebound CO at the time t after the flash-off. to and

¹¹

are temperature dependent parameters. The simplest way to explain this non- exponential behavior is to assume that the binding process in this low-temperature regime is governed by an activation energy, E, that is not sharp. If g ( E ) denotes the probability of having a Mb molecule with activation energy between E and E + dE, H ( t ) is given by

where the constant 3, is related to the activation energy E and the attempt factor A by tlie Arrlienius equation,

1

⁼

A

e - E l k T ,

( 3 ) With eq. (3), the time derivative of H can be written as

The attempt factor A is usually very large, tlie upper limit of the integral can be replaced by infinity, and 2(t) becomes the Laplace transform of g ( i )

:

From a measured d(t), the energy distribution func- tion can be obtained by the inverse Laplace transfor- mation. As an example, relation (I) for H(r) leads to the distribution function

The peak of this distribution ( d g / d i

=

0 ) occurs at

With eq. (3) we write

By plotting k T l n A,,,, versus T, A and E,,,, can be determined. With A known,

goL)

can be transformed into g(E).

We have determined g ( E ) for the low-temperature reaction for two heme proteins, Mb and cytochrome P 450. The peak energy is about 80 meV for Mb and 35 meV for cytochrome P 450

;

tlie first distribution has a width of about 50 meV, tlie second of about 25 meV. The appearance of energy spectra rather than discrete energies in these protein reactions is unex- pected. Tlie cause is not yet clear. Two possibilities must be explored

:

(I) The biomolecules are so sensitive tliat inhomo- geneities in the surrounding afrect tlie activation ener- gies. At the temperature

a1

which tlie relation (1) is valid, the glycerol-water mixture has beconie a glass and some inhomogeneities are be to expected.

(2) During the folding process, it is possible that a given priniary structure results in a group of tertiary structures. Myoglobin then, for example, would not have just one well-defined struct~lre but a number of very similar ones, sometimes called conformers or peers [4]. Tlie various conformel-s would have slightly different activation cnergies. At the present time it is not clear which explanation (if either) is correct.

Another question raised by these measurements refers to the physical interpretation of the activated processes. What gives rise to a n activation enerfy of the order of 0.1 eV or less

?

It is possible that a detail- ed comparison of tlie energy levels of iron in heme proteins with more measurements of tlie typc described here will provide a n answer. Tlie energy levels of iron can be obtained from measurements of tlie tempera- ture dependence of tlie quadrupole splitting using tlie Mossbauer effect [5]. It is interesting to note tliat Mossbauer techniques and kinetic experiments tli~ls possibly will meet.

In any case, the experiments discussed here denions- trate that reactions in biolnolecules below the physiolo- gical temperature range lead to additional information.

Before the full impact of tlie new results can be assessed, more measurements and additional interpretation are needed.

References

[I] DICKERSON, R . E. and GEIS, I., The Strttct~tre ottd Actiotl of [3] AUSTIN, R. H., BEESON, K., EISENSTEIN, L., FRAUENFI:LIIER, Proteirls. (Harper and Row, New York) 1969. H., GUNSALUS, I. C . and MARSI-IALL, V. P., Scic,ttc,r MCLACHLAN, A. D., Attt~. Rev. P1ty.r. C / I O I ) I . 23 (1972) 165. 181 (1973) 541.

121 WEISSBLUTH, M., The Physics of Hemoglobin, in Stt.rtctrtrc

~ ~ ~ ~ r f i r ~ ~ , vol. 2 (springer verlag, ~ ~ ~1967, i i1 , ~ [41 KLOTZ, , 1. M., Arch. Biotllenr. Bio~l?l's. 116 1196'5) 92.

ANTONINI, E. and BRUNORI, M., H~t)loglobi,t nlld Mj.og/obil, [51 T ~ < A U T W E ~ N . A,, EICHCR, H., MAYER, * L F S E ~ \ ' , " A . ~ it2 Their Reuctiotls ~ ~ * i t h Ligot~cls. (North-Holland, WAKS, M . , ROSA, J . and BEUZARD, Y., J. C l ~ c ~ ~ t t . Plr~'.r.

Amsterdam) 1971. 53 (1970) 963.