Download ELECTRON TRANSFER PATHWAYS IN BLUE COPPER

2nd INTERNATIONAL CONFERENCE ON BIOINORGANIC CHEMISTRY
SL27 — TH
I. PECHT
O. FARVER
A. LICHT
Department of Chemical Immunology
Rehovot 76100
Israel
ELECTRON TRANSFER PATHWAYS
IN BLUE COPPER PROTEINS
Electron transfer reactions play a central role in
biological energy conversion processes. The mechanisms employed by the different protein elements of these systems are now being examined at
the resolution of amino acid residues involved in
the reactions. Thus, specific loci on the protein's
surface involved in electron-transfer and the media separating them from the metal ion active centers are investigated [1,2]. An affinity labeling
procedure for such loci has been developed,
taking advantage of the chemical properties of the
Cr(II)/(III) couple: While Cr(II) ions are exceptionally strong reductants and exchange their ligands
very fast, the Cr(III) ion exchanges its ligands rather slowly [3]. Thus, Cr(II) can coordinate to one
or more amino acid residues of the protein while
transferring to its active center an electron. Upon
oxidation to Cr(III), it maintains the same liganding residue(s) in its coordination sphere. Hence
identification of the attachment site of the Cr(III)
may provide information about the electron transfer locus. The Cr(III) binding sites are determined
primarily by proteolytic cleavage of the different
labeled proteins [4-7]. In some cases spectroscopic
methods have been useful in corroborating these
assignments [8].
Several copper proteins have been examined by
the above approach. These include: The blue copper containing electron carrier proteins — azurins
from several bacteria (Ps. aeruginosa [4] and
Alc. Faecalis). Plastocyanin (French bean) the
electron mediator of the photosynthetic apparatus
Rev. Port. Quím., 27 (1985)
[5]. Stellacyanin [6] and the oxidase laccase isolated from the Japanese lacquer tree (Rhus vernicifera). Our prime interest focused on the spatial relation between the copper site and the locus of
Cr(III) labeling and predominantly on the role of
the labelled regions in the functions of the examined proteins.
In Ps. azurin, Cr(III) labeling of the neighborhood of His-35 imidazole has been found [4].
This amino acid residue has been implicated
earlier in the electron-transfer function of the protein. Even anionic inorganic redox partners seem
to react at this region [8]. In stellacyanin, the site
of the single Cr(III) bound to the protein is currently being identified.
The cuprous ions in the Cr(III) labeled plastocyanin, azurin and stellacyanin can be fully reoxidized by inorganic or enzymatic agents without any
loss of the bound chromium. The single and the
same Cr(III) ion originally coordinated to azurin
and stellacyanin remains bound through several
Cr(II) reduction and reoxidation cycles. In contrast, one can label plastocyanin (French bean)
with several Cr(III) ions by repeated redox cycles.
As illustrated in the table up to 6.0 Cr(III) ions
can be bound to one plastocyanin molecule in seven reduction steps. Reoxidation has been attained in these cycles with Co(III) dipicolinate and
analysis of the protein's copper content established the stoichiometry.
Cr(II) Labeling Upon Multiple Reduction-Oxidation Titrations of Pc
TITR.
I
II
1E
13Z
Y
II
Cr /Pc
0.89
1.7
2.5
3.5
4.4
5.2
YE
-
6.0
The numbers represent moles of Cr(II) bound to
plastocyanin after each cycle (Roman numbers).
The labeled proteins now carry the Cr(III) modification on their surface. To examine the physiological significance of the labeled sites, the reactivities of native and singly Cr(III) labeled forms of
azurin and plastocyanin were compared. It became apparent that the Cr(III) label attenuated the
reactivity of both azurin and plastocyanin [9,10]
with only one of their respective two partners.
This led to the conclusions that on both proteins:
45
SESSION LECTURES
a) There are probably two distinct and physiologically operative electron transfer sites. b) One of
these sites is centered around the respective Cr(III)
labeled region. c) By elimination, the second is at
the exposed, homologous imidazole of His-87 or
117 in Pc and Az, respectively.
REFERENCES
111 O. FARVER, I. PECHT, in R. LONTIE (ed.), «Copper Proteins and Copper Enzymes», CRC Press, vol. I, 1983, pp.
187-214.
[2] R.A. MARCUS, N. SUTIN, Biochim. Biophys. Acta (1985),
in press.
[3] H. TAUBE, Science, 226, 1028-1036 (1984).
[4] 0. FARVER, I. PECHT, Isr. J. Chem., 21, 13-17 (1981).
[5] 0. FARVER, I. PECHT, Proc. Nat!. Acad. Sci. USA, 78,
4190-4193 (1981).
16] G. MORPURGO, I. PECHT, Biochem. Biophys. Res. Commun., 104, 1592-1596 (1982).
[7] G.Q. JONES, M.T. WILSON, J. Inorg. Biochem., 21,
159-168 (1983).
[8] V.C. CHO, D.F. BLAIR, U. BANERJEE, J.J. HOPFIELD,
H.B. GRAY, I. PECHT, S.I. CHAN, Biochemistry, 23,
1858-1901.
[9] O. FARVER, Y. SHAHAK, 1. PECHT, Biochemistry, 21,
1885-1890 (1982).
[10] 0. FARVER, Y. BLATT, 1. PECHT, Biochemistry, 21, 3556-3561 (1982).
SL28 — TH
K. LERCH
active site copper. Met- and oxytyrosinase contain
two tetragonal Cu(II) ions antiferromagnetically
coupled through an endogenous bridge with the
exogenous oxygen molecule in oxytyrosinase
bound as peroxide. In halfmettyrosinase the two
coppers are present in a mixed-valence state [2].
The chemical and spectroscopic properties of the
different forms are surprisingly similar to those
reported for the oxygen-binding hemocyanins [3].
However, differences between the tyrosinase and
hemocyanin active sites are apparent from peroxide displacement and binding studies of tyrosinase
substrate analogues [4]. Binding of L-mimosine
and various derivatives of benzoic acid to halfmettyrosinase results in very unusual Cu(II) spectral features. They relate to a significant distortion of the Cu(II) site as shown by the rhombic
splittings and perpendicular hyperfine structure of
the EPR spectra [5]. In addition, these competitive inhibitors are found to bind to the enzyme with
an equilibrium constant higher by one order of
magnitude relative to aqueous Cu(II) [6]. It is suggested that the protein environment of the binuclear copper complex contributes significantly to
the stabilization of substrate analogues binding to
the active site. This stabilization and the concomitant change from a tetragonal toward a trigonal
bipyramidal geometry of the Cu(II) site seem to
greatly assist the catalytic hydroxylation reaction
of tyrosinase. It is proposed that the binding of a
monophenol substrate to oxytyrosinase leads to a
distortion of the Cu(II) complex, thus labilizing
the peroxide. This then leaves a reactive, polarized
peroxide which in turn can hydroxylate the monophenol most likely via an electrophilic attack on
the aromatic ring.
Biochemisches Institut der Universitàl Zürich
Winterthurerstrasse 190, CH-8057 Zürich
Switzerland
THE BINUCLEAR COPPER CENTER
AND THE REACTION MECHANISM
OF TYROSINASE
Tyrosinase is a copper-containing monooxygenase
catalyzing the formation of dark colored melanin
pigments [1]. Various forms of the enzyme can be
obtained (met-, halfmet-, oxy- and desoxytyrosinase), depending on the oxidation state of the
46
REFERENCES
[1] K. LERCH, in H. SIGEL (ed.), «Metal Ions in Biological
Systems», vol. 13, Marcel Dekker, New York, 1981, pp.
143-186.
[2] K. LERCH, Mol. Cell Biochem., 52, 125-138 (1983).
13] E.I. SOLOMON, in T.G. SPIRO (ed.), «Copper Proteins»,
Wiley and Sons, Inc., New York, 1981, pp. 41-108.
[4] R.S. HIMMELWRIGHT, N.C. EICKMAN, C.D. LUBIEN, K.
LERCH, E.I. SOLOMON, J. Am. Chem. Soc., 102, 7339-7344
(1980).
[5] M.E. WINKLER, K. LERCH, E.I. SOLOMON, J. Am. Chem.
Soc., 103, 7001-7003 (1981).
[6] D.E. WILCOX, A.G. PORRAS, Y.T. HWANG, K. LERCH,
M.E. WINKLER, E.I. SOLOMON, submitted for publication.
Rev. Port. Quim.,
27 (1985)