2nd INTERNATIONAL CONFERENCE ON BIOINORGANIC CHEMISTRY
100 G
].ti
PS1.22 — TH
A
TINY VAN HOUWELINGEN
G.W. CANTERS
J.A. DUINE
a
J. FRANK, JZN.
G. STOBBELAAR
Department of Chemistry
Gorlaeus Laboratories
State University Leiden
P.O. Box 9502, 2300 RA Leiden
The Netherlands
Fig. 4
(A) EPR spectra of B12 containing protein from M. barkeri
(DSM 800); (B) EPR spectra of the extracted corrinoid from
cells of M. barkeri (DSM 800).
Both methyl
B 12
and
Biochemical Laboratory
Technical University Delft
Julianalaan 67-67a, 2628 BC, Delft
The Netherlands
protein and methyl extracted corrinoids were
photolyzed under reducing conditions.
EPR Conditions: Microwave frequency 9.28 GHz; Temperature 77 K; Microwave power 20 mW; Field modulation 2 mT;
Gain 8 x 10 4
ted to the cobalt both in its bound form to the
protein and in the free form.
M. barkeri is until now the only methanogen where the presence of a B12 protein was reported
[2,5]. In these bacteria it was shown that factor
III is the most abundant corrinoid in the cells and
that it is also the corrinoid associated with the B12
protein.
Although the physiological role of these proteins
is not yet clearly established, they seem to be involved in the biosynthesis of CH 3 S-CoM or CH 4
from CH 3 OH [2].
ACKNOWLEDGEMENTS
Work supported by INIC, JN/CT, U.S.A.I.D. and Quatrum.
REFERENCES
[1] J.G. ZEIKUS, Bact. Rev., 41, 515-541 (1977).
[2] J.M. WOOD, I. MOURA, J.J.G. MOURA, H. SANTOS, A.V.
XAVIER,
J.
LEGALL, M. SCANDELLARI, Science,
216, 303-
-305 (1982).
[3] K.BERNHAUER, G. WILHARM, Arch. Biochem. Biophys.,
83, 248 (1959).
[4] O.D.
HENSENS,
H.A.O.
in D.
HILL,
C.E.
MCCLELLAND,
R.J.P.
(ed.), «B 12 », vol. 1, Wiley,
New York, 1092, pp. 463-500.
[5] P. VAN DER MEIJDEN, H.J. HEYTHUYSEN, A. POWELS,
F.P. HOWEN, C. VAN DER DRIFT, G.D. VOGLY, Arch. MiWILLIAMS,
DOLPHINS
crobiol., 134, 238-242 (1983).
Rev. Port. QuIm., 27 (1985)
A BLUE COPPER PROTEIN
FROM THIOBACILLUS VERSUTUS
The multifarious coordinating capabilities of copper are reflected by the variety of metalloproteins
in which Cu occupies the catalytically active site.
The usual classification of copper proteins distinguishes between three or four types [1].
As more and more copper proteins are discovered
and characterised, it is becoming clear that within
each class diversity reigns. For instance, for the
type I blue copper proteins, the most extensively
studied class up till now, it has been found that
redox potentials may vary from 180 to 760 mV,
molecular weights from 10 to 20 kD and pI points
from 4 to 11. Most intriguing is the coordination
of the Cu. It has been demonstrated by crystallographic techniques in a number of cases that the
metal is surrounded in a distorted tetrahedral
fashion by an N 2 SS* coordination [2-4]. The
nitrogens are provided by two histidines and the
sulfurs derive from a methionine and a cysteine.
However, stellacyanin lacks methionine and Russian researchers have reported a blue copper protein which does not seem to contain cysteine [5,6].
It is not understood how the details of the Cu
coordination relate to the spectroscopic properties
and the redox potential of the protein and further
structural studies and a search for new type I copper proteins are needed.
177
POSTER SESSIONS: I. METALLOPROTEINS
Here the isolation of a blue copper protein from
Thiobacillus versutus (previously called Thiobacillus sp. strain A2, see [7]) grown on methylamine
is reported. This protein is part of a redox chain
that consists of probably 4 proteins and that takes
care of the conversion of methylamine into the
aldehyde. The primary enzyme is a methylamine
dehydrogenase (MADH) of which the prosthetic
group is a pyrrolo-quinoline quinone (PQQ) [8].
The next two links in the chain are the blue copper protein mentioned above and a cytochrome,
followed probably by a final oxidase. Isolation
and purification of the blue copper protein will be
described on the poster. A detailed characterization is nearly completed (ESR, NMR, optical
spectra, redox potential, pI, molecular weight,
etc.) and the results will be reported as well. The
data obtained so far (E o = 256 mV,
maximum of the visible absorption band at 596
nm, type I ESR spectrum, MW = 12,000 - 13,000)
justify the conclusion that the protein is an amicyanin type blue copper protein [9].
REFERENCES
[1] E.T. ADMAN, in PAULINE HARRISON (ed.), «Topics in
Molecular and Structural Biology», MacMillan.
[2] P.M. COLMAN, H.C. FREEMAN, J.M. Guss, M. MURATA,
V.A. NORRIS, J.A.M. RAN/SHAW, M.P. VENKATAPPA,
Nature, 272, 319 (1978).
[3] E.T. ADMAN, R.E. STENKAMP, L.C. SIEKER, L.H. JENSEN,
J. Mot. Biol., 123, 35 (1978).
[4] G.E. NORRIS, B.F. ANDERSON, E.N. BAKER, J. Mol. Biol.,
165, 501 (1983).
[5] C. BERGMAN, E.K. GANVIK, P.O. NYMAN, L. STRID,
Biochem. Biophys. Res. Commun., 77, 1052 (1977).
[6] V.T. AIKAZYAN, R.M. NALBANDYAN, Biochim. Biophys.
Acta, 677, 421 (1981).
[7] A.P. HARRISON JR., Intern. J. System. Bacteriol., 33, 211
(1983).
[8] J.A. DUINE, J. FRANK JZN., Trends Biochem. Sci., 6,
278 (1981).
[9] J. TOBARI, Y. HARADA, Biochem. Biophys. Res.
Commun., 101, 502 (1981).
178
PS1.23 — TH
M.C. FEITERS
Chemistry Department
University of Manchester
U.K.
and
Daresbury Laboratory
Warrington
U.K.
C.M. GROENEVELD
G.W. CANTERS
Department of Chemistry
University of Leiden
The Netherlands
S.S.
HASNAIN
Daresbury Laboratory
Warrington
U.K.
EXAFS STUDIES ON OXIDIZED
AND REDUCED AZURIN
AT HIGH AND LOW pH
X-ray fluorescence spectra [1] at the Cu K-edge
were taken of the blue copper protein, azurin,
from Pseudomonas aeruginosa, in reduced and
oxidized state, and at high and low pH, to assess
the effect of these conditions on the coordination
sphere of the copper.
There is no change in either edge position or
XANES (X-ray absorption near edge structure)
when the pH is changed. However, there are
changes in the XANES upon oxidation or reduction at both high and low pH. The edge position
of the oxidized protein is at higher energy than
that of the reduced protein. This is in agreement
with what is expected because of the valence change of the copper.
The EXAFS (extended X-ray absorption fine
structure) of oxidized protein is strongly reminiscent of that of lyophilized azurin [2]. There is almost no change when the pH is altered; the major
shells can be fitted with the same set of parameters, involving 2 N atoms and 1 S atom, from
His-46, His-117 and Cys-112, respectively, except
for a small correction in E o .
There are pronounced changes upon reduction:
Rev. Port. Quint., 27 (1985)