Download Juxtaposition of particular amino acid residues may contribute to the

1098
BIOCHEMICAL SOCIETY TRANSACTIONS
Juxtaposition of particular amino acid residues may contribute to the protection of proteins from
active free radicals
DAVID GRANT, WILLIAM F. LONG and
FRANK B. WILLIAMSON
Department of Biochemistry, University of Aberdeen,
Marischal College, Aberdeen, AB9 IAS, Scotland, U.K.
The production of species of oxygen free radicals is a normal
consequence of aerobic metabolism, and is also associated
with specialized activities such as phagocytosis by macrophages and polymorphonuclear leukocytes. The presence of
abnormally high concentrations of free radicals, however,
appears to be associated with tissue ageing, and with various
disease states, particularly neoplasia. Intracellular concentrations of free radicals may be ordinarily governed by
the activity of the enzymes superoxide dismutase, catalase,
and glutathione peroxidase. Other naturally occurring
antioxidants include low-molecular-mass thiol-groupcontaining compounds, vitamins C and E, fi-carotene and
urate. In addition, we suggest that many of those proteins
which function in extracellular environments potentially
rich in oxygen free radicals may carry an inbuilt antioxidant
system.
To act as an effective free radical sink, a molecular species
would need to be able to accommodate delocalized unpaired
electrons without undergoing such molecular rearrangements as would immediately lead to degradation of the
species, and then, perhaps by steric hindrance o r electrostatic repulsion, to prevent the close approach of further
potential acceptors of the electrons. The juxtaposed sidechains of particular amino acid residues in proteins exhibit
the kinds of structural features which might enable these
functions to be performed.
We predict that unpaired electrons of active free radicals
might be readily accommodated in a delocalized form over
the aromatic rings of phenylalanine, tryptophan, tyrosine
and perhaps histidine residues. Tyrosine residues might be
especially effective free radical sinks, since a dissociable
phenolic hydroxyl group would form a suitably receptive
site for unpaired electrons, which would then be partially
shared with the ring carbons. In another context (Reichard
& Ehrenberg, 1983), the stability of the tyrosine residue free
radical has been discussed, and a Cu2+chelate with tyrosyl
tyrosine has superoxide dismutase activity (Brigelius et al.,
1979, the cation perhaps finally accepting an electron
initially interacting with the hydroxyl group(s). A putative
free radical scavenging role for tyrosine residues could
presumably be modulated by phosphorylation or sulphation.
We interpret the reported inhibition of macrophagemediated tumoricidal activity by neuropeptides and neurohormones (Koff & Dunegan, 1985) in terms of a blockage of
the macrophage oxidant activity. The only common feature
in inhibitory molecules of otherwise disparate structures is
a plethora of hydroxyl groups.
Another possible target for unpaired electrons comprises
those residues containing bivalent sulphur atoms, i.e.
residues of methionine, cysteine and the disulphide bridges
of cystines. Those structures in proteins in which sulphur
atoms are closely clustered would be particularly susceptible. Such frameworks might, like the polyhedral arrange-
ments of sulphur atoms in several inorganic compounds, be
stabilized by transannular bonding involving delocalized
electrons shared between formally bivalent sulphurs. A
protein so oxidized, however, might be functionally
impaired, and such a situation could be ameliorated by
juxtaposition of sulphur clusters with more stable free
radical sinks, particularly, perhaps, the tyrosine residues
mentioned above. An analogous situation to the synergistic
effectiveness of hindered phenols and sulphur-containing
antioxidants as free radical scavengers in polypropylene and
rubber production would then exist. It is possible that,
intracellularly, interaction between vitamin E, which is a
hindered phenol, and glutathione, might lead to similar
synergism.
Features of several extracellular protein structures seem
to accord with these ideas. Batteries of disulphide bridges
are often found in close proximity to residues of tyrosine.
This relationship is readily discernible in three-dimensional
representations of such proteins, and also occasionally, as in
the disulphide knot system of fibrinogen, and in kringle
structures, directly from primary structures. Extracellular
proteins of equivalent functional status, but lacking such
organization, for example human a, -proteinase inhibitor,
are particularly susceptible to damage by free radicals
(Matheson el al., 1979). In this latter case, free radical
interaction occurs at the reactive centre methionine residue
358, which appears to be unprotected by closely approaching aromatic side-chains. For serine proteinases, and several
other groups of extracellular proteins, closely approaching
aromatic/divalent sulphur side-chains, particularly tyrosine/
cysteine combinations, are evident from primary structures,
and more especially, from three-dimensional representations.
Experimental evidence which accords generally with
the above ideas comes from a study of the interaction of
very reactive, hydrophobic, photogenerated, radiolabelled
nitrenes with the ovine membrane protein opsin (Davidson
& Findlay, 1986). The principal targets for interaction with
these short-lived reactive intermediates, which may be
regarded as analogous to the most reactive of the naturally
occurring oxygen free radicals, are a tyrosine residue (43)
adjacent to a methionine residue, and a cysteine/tyrosine
pair (residues 222, 223). Our necessarily tentative suggestions may be explored by equivalent experiments in other
protein systems, coupled to detailed examination of threedimensional molecular structures.
We thank the Cancer Research Campaign for grants which supported
work in Dr Long and Dr Williamson's laboratory during the preparation of this communication.
Brigelius, R., Hartmann, H.-J., Bors, W., Saran, M., Lengfelder, E. &
Weser, U. (1975) Hoppe-Seyler's Z . Physiol. Chem. 356, 739--745
Davidson, M. D. & Findlay, J. B. C. (1986) Biochem. J . 236, 389-395
Koff, W. C. & Dunegan, M. A. (1985) J . Immunol. 135, 350-354
Matheson, N. R., Wong, P. S . & Travis, J. (1979) Biochem. Biophys.
Res. Commun. 88, 402-409
Reichard, P. & Ehrenberg, A. (1983) Science 221, 514-519
Received 15 June 1986
1987