Vol. 51, No. 4,1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MAGNETICCIRCULARDICHROISMOF NON-HEME IRONPROTEINS* D. D. Ulmer', B. Holmquist and B. L. Vallee+ The Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts
Received
March
5,
1973
The magnetic circular dichroism (MCD) at 45 kgauss has been determined for a group of non-heme iron proteins. Both transferrin and conalbumin exhibit a single, positive ellipticity band at 330 nm ferre([@I = 560). Oxy- and methemerythrin, spinach and clostridial dox&s and rubredoxin all display distinctive multibanded spectra which may reflect such factors as coordination of the metal, its ligands, metal bridging by other atoms, and varying degrees of metalmetal coupling. The MCDspectra of both ferredoxins and rubredoxin undergo dramatic change upon oxidoreduction providing a potential means for relating the electronic structure of the iron to protein In contrast to the plant ferredoxins, the magnetic field function. does not significantly affect the CD spectra of adrenodoxin and putidaredoxin. Studies of the extrinsic
Cotton
effects
of metalloproteins
circular
dichroism (CD) have contributed
importantly
of their
conformations and metal-binding
sites
by
to the understanding
(l-3).
Whereas conventional
CD signals arise only from asymmetric chromophores, imposition of a properly oriented magnetic field,
MCD, can induce optical
Effect
-- in virtually
origin
of the Faraday Effect
distinct
all
electronic
transitions.
activity
-- the Faraday
Since the physical
and that of the Cotton Effect
are quite
(4,5) the former would be expected to provide further
into the electronic
structure
To assess the potential
insight
of the metal sites of metalloproteins. usefulness of magnetic circular
dichroism
@CD), we have examined a group of non-heme iron proteins which have been characterized
previously
by conventional
*
CD and resonance spectral methods.
This research was supported by Grants GM-15003 and GM-02123 from the National Institutes of Health of the Department of Health, Education and Welfare. tPresent address: Martin Luther King, Jr. Hospital, Department of Medicine, Los Angeles, California. + To whominquiries should be addressed. Copyright 0 I9 73 by Academic Press, Inc. AN rights of reproduction in any form reserved.
1054
BIOCHEMICAL
Vol. 51, No. 4,1973
A preliminary proteins
report
has been Metal
METHODS:
given
(Sigma
and measured
in
was diluted
work
of human serum
Chemical
to 1 mg/ml
in
0.15
a gift
Lovenberg,
a gift
of Dr.
to remove
pH 8.0, Dr.
'T. Kimura,
prior
ethanol
Drs.
in 0.05
M Tris-Cl,
Chemical
Co.)
several
days
to remove
MCD spectra a Varian
was dialyzed
were
Model
measured
of 40 to 45 kgauss. dispersion experimental MCD is NM for
conditions
expressed
is
calculated
appropriate
solvent
N2 prior
oxidase,
61
CD spectra absence
were
from butterfat
pH 8.5,
for
All
equipped
at field to permit
of a magnetic
CD.
instrument
measured
cm2 decimole
conventional
CD
solenoid
was adjusted
as [O]M in degrees as for
liquid
by
sulfate.
width
in the
under
of
just
prepared
M Tris-Cl,
on a Cary Model
The slit
a gift
pH 7.49,
generously
Xanthine
superconducting
of 2 nm or less.
phosphate,
0.01
Hemerythrin,
adrenodoxin,
was stored
versus
ammonium
V-4145
while
pH 7.4.
The
0.1 M Tris-cacodylate,
Putidaredoxin,
C. Gunsalus,
also
pH 7.3.
was 2.55.
against
M sodium
Lovenberg,
rubredoxin,
material
salt,
in 0.01
and I.
(Sigma
with
and excess
measurements.
J. L. Lipscomb
to measurement
nm of this
type to measure-
Walter
M Tris-Cl,
Co.)
previously
pH 7.3,prior
by Dr.
in 0.15
Chemical
ferredoxin,
and -~ Clostridial
was dialyzed
was dissolved
to spectral
provided
was dissolved
Klotz,
Spinach
M NaCl,
pH 7.28
at 2801490
Irving
substituted
as described
pH 8.5.
kindly
on 0.1 41 T&s-Cl,
of absorption
of Co(II)
(Hoechst
prepared
M Tris-0.8
ferredoxin,
of Dr.
studies
transferrin
were
buffer,
was dissolved
ratio
Co.)
0.1 M Tris-Cl
Clostridial
and related
RESEARCH COMMUNICATIONS
(6,7).
complexes
and conalbumin
ment.
of this
AND BlOPHYSiCAL
-1
under
strengths
spectral identical
field. kilogauss
samples
-1
were
, where
corrected
blanks.
RESULTS: Figure broad
negative
1 compares
the CD and
CD ellipticity
band
MCD
spectra
centered
1055
of iron
transferrin.
at 465 nm coincides
A precisely
(1) III,
Vol. 51, No. 4,1973
with
the Xmax of the absorption
band at the
BIOCHEMICAL
330 nm.
A magnetic
330 nm band but
not
spectrum; field
that
AND BIOPHYSJCAL RESEARCH COMMUNlCATiONS
there
is
1. CD spectra presence ( of transferrin ( text.
at 465 nm (Figure
1).
between
measurements
field
(Figure
1, insert),
([O],
= 560).
This
is
not
altered
though
temperature to that
2 a-c
and of a bacterial per
mole
(Figure shows
thought
are
similar,
very
exhibits
three
but distinct
the
om
presence
a B-term
in temperature
from
of a C-term
requires
of a magnetic
signal
at 330 nm
since
its
and +30"
the
intensity
C (See below),
examination
is virtually
of a wider identical
1, insert). of hemerythrin,
of the protein
(8).
MCD spectra
from
Hemerythrin
ferredoxin. identical
positive
-30
of conalbumin
to have nearly
their
and absence
represents
the MCD spectra
and a plant
and met-forms
enhances
The MCD spectrum,
sharp
The MCD spectrum
the oxy-
markedly
as a single,
likely
elimination
of transferrin
Figure
atoms
transition
range.
in the
manifests
by variations
definitive
positive
of human serum iron transferrin in the absence ( ..a* ) ) of a 47 kgauss magnetic field. INSERT: MCD spectra ) and conalbumin ( - - - ). For conditions see the
and
difference
a sharper,
of up to 47 kilogauss
WAVELENGTH,
Fig.
also
differ
MCD bands,
1056
contains
electronic The CD spectra significantly: at 330,
sipunculid
worms,
two iron
structures of the
in both two forms
oxyhemerythrin
380 and 503 nm, superimposed
Vol. 51, No. 4,1973
BIOCHEMKAL
AND EtOPHYSlCAl
RESEARCH COMMUNICATIONS
400 500 WAVELENGTH, nm
300
600
of a) oxyhemerythrin ( ) and methemerythrin Fig. 2. MCD spectra ( - - - ); of b) oxidized > and reduced ( - - - ) clostridial ( ferredoxin; and c) of oxidized ( ) and reduced ( - - - ) spinach ferredoxin. For conditions see text.
upon a broad (Figure Jith
envelope
2a).
of positive
In contrast,
a positive
ellipticity
methemerythrin
increasing generates
peak at 438 nm, and a negative
to be a highly
discriminative
sensor
of the
with
a broad
trough
at
electronic
decreasing biphasic
wavelengt
band
350 nm.
MCD appears
properties
of the
oxy-
and met-forms. A high absorption
magnetic
field
or conventional
(Figure
2 b),
plified
by the oxidized
ellipticity doxins
that
bands
over
MCD may serve
properties.
spinach and above
Both
yet
not
observed
Clostridial
(2 g at Fe/mole)
protein
which
those
transfer
additional
to correlate
details of oxidized
ferredoxin
in electron
resolves
resolves
CD spectra
and spinach
function
dithionite
also
seen in and their
transitions, their
the MCD and CD spectra
structural
seven
2c) as exemdistinct
the CD spectrum. reduction
with
(Figures with
of xanthine
1057
(8 g at Fe/mole)
(Figure
exhibits
in the
The
ferre-
sodium
Zb,c)
suggesting
pertinent oxidase
new
functional (9)
closely
Vol. 5 1, No. 4, 1973
resemble
those
analogous
further
Above
ferredoxin.
The implication
structures
of both
intense
bands.
390 nm, may represent particularly
these
RESEARCH COMMUNlCATfONS
non-heme
that
this
reflects
iron
proteins
study.
300 nm the MCD of oxidized
overlapping,
Reduction
AND BIOPHYSICAL
of spinach
electronic
requires
BIOCHEMICAL
markedly
A biphasic
an A-term
helpful
312 and 336 nm (Figure
3) (see
the environment
the negative
3) identified
manifests
component,
(Figure
in defining enhances
rubredoxin
as an envelope
centered
between
discussion), the single
and positive
ellipticity
previously
in
380-
and could
of
of
iron
be
atom. bands
the CD spectrum
at
of
rubredoxin.
of oxidized Fig. 3. MCD spectra For conditions see the text.
Adrenodoxin, strengths supplied
(Kimura, by Dr.
to 45 kilogauss, plant visible
in accord
I.
Gunsalus, indicating
ferredoxins, and near
DISCUSSION:
personal
both
with
these
ultraviolet
characteristic
) and reduced
earlier
observations
communication), do not that
Zeeman splitting
an atom generates
( -
iron
at lower
field
and putidaredoxin,
exhibit
in contrast
proteins
( - - - ) rubredoxin.
lack
appreciable
kindly
MCD at fields
to the more negative significant
degeneracy
up
potential in
the
transitions.
of degenerate MCD signals
1058
ground
or excited
categorized
states
in accord
of with
Vol. 51, No. 4, 1973
their
origins
and differentiated
dependencies. are
BIOCHEMICAL
C terms
temperature
by means of their
are monophasic
dependent,
the
phasic
and temperature-independent
mixing
of non-degenerate
dipole
polarization
characteristics
e.g.
present
is
as well
as the
As concerns
ligands
the
Transferrin octahedrally metals
coordinated
are not
complexes, in time
their
and conalbumin
thought
binding resonance
each of the
The virtually
two irons
identical,
prove
and CD spectra, of overlaps.
The
to simpler
systems,
compared
MCD spectra
doubtless
the valence
of the metal,
is
by other
coordination
atoms,
geometries.
of cobalt
suggesting
two high and phenolic
(12).
two iron
atoms
spin
II
those
complexes
of some
Fe (1II)atoms
oxygen
Based on the seems identical
have been
interpreted
may exist
in a different
single
absorption
the MCD spectra
contain
to interact
signals
should
molecules,
(6).
to nitrogen
of the
it
metal-bridging
geometries
metalloenzymes
of more complex
atoms and their
found
of particular
substituted
iron
interaction,
we have
calculations.
empirical.
including
iron
theoretical
or because
much more
of factors
latter,
in
magnetic
to spectroscopic
However,
though
of the non-heme
of the
for
probabilities
of metal-metal
to be characteristic Co(I1)
electric
and of polarization
analysis
concealed
remains
by a variety degrees
the
such expectations,
diversity
varying
are monoinduced
reflect
can lead
basis
in
transitions
interpretation
influenced
These
as yet uncertain.
support
The rich
which
of different
states
a quantitative
of low transition
studies
spectral
the former
field
molecules
electronic
transitions.
in delineating because
B terms,
states
chromophoric
MCD can assist
metalloproteins,
either
are biphasic;
from magnetic
and excited
of their
provide
to which
valuable
result
of simple
of their which
The degree
not.
and temperature
(5).
properties
assignments
are
RESEARCH COMMUNICATIONS
patterns
and A terms
latter
ground
The MCD spectra and symmetry
AND BIOPHYSICAL
transition
1059
in
ligands
(11).
stability
differences
that
conformation
the MCD spectra
The
of their
though
to suggest
(10)
at any given (13,14).
of the
two
Vol. 5 1, No. 4, 1973
proteins
BIOCHEMICAL
(Figure
to oxygen
1) may be characteristic
significantly
features
of all
of these
coupling
of their
metal
regard
positions that
the
transitions is
and oxidized
thought
of hemerythrin
their
(17)
to form
accounting
perhaps
for
the differences
in nearly
those
spectra.
indicate
may respond
between likely
In
the same spectral ferredoxin
proteins
might
degrees
distinctive
a bridge
and sulfur
electronic
with
Clostridial
in both
ferredoxins
varying
together
bands
iron
the
to reflect
for
ellipticity
and plant
However,
taken
account
of the
Oxygen
closely
play
features
to the adjacent
an analogous of their
role
MCD which
similar. The sulfur
three
positive
(Fig.
2 c).
bound
However,
ferromagnetic studies
both
the iron
that
investigation
spectrum other
apparent
additional
ultraviolet
coupling
between
forms
their
oxidized
of these
of these
of the oxidized non-heme A-term
iron
centered
noted maxima
in each.
proteins
from
distinguish anti-
by NMR
ferredoxins
It
above
strong
as suggested
and from
exhibit
differ
each other,
suggesting
may be anticipated
different
sources
may
variations.
of cysteine spin
centers,
and bacterial
environment
also
due to unusually
equivalents
Fe atom of oxidized
and high
and visible
metal
of plant
ferredoxin,
of the bands
perhaps
protein,
of a series
groups
spinach
the position
atom has a unique
The single
Fe(III)
of oxidized
at or near
the plant
from
the basis
sulfhydryl
atoms
The reduced
that
clarify
of
yet
exchange
(15).
strikingly
iron
MCD maxima
the MCD spectrum
the
16) which,
positive
in some ferredoxins,
spin
15,
2, a-c).
are believed
oxyhemerythrin
field. atoms
(Fig.
may likely
three
of both
magnetic
are
Fe (11% coordinated
spin
and the bacterial
proteins
(8,
ligands,
analogous
metal
of hemerythrin more complex
of iron-iron
this
of high
and nitrogen.
The MCD spectra are
AND BIOPHYSJCAL RESEARCH COMMUNICATIONS
and reduced
in a distorted Fe (II)
protein proteins between
tetrahedron,
states, is here
rubredoxin,
respectively
unusually
intense
examined,
380-390
1060
nm (Fig.
coordinated is
in
the high The MCD
(18,19). compared
and its
bands
3),
in marked
with
to those are
split
contrast
of with with
an
Vol. 51, No. 4,1973
BlOCHEMtCAL
the high spin iron in tranaferrin
AND BiOPHYSlCAL
RESEARCH COMMUNICATIONS
and conalbumin (Fig. 1).
The intense, bi-
phaaic signal of reduced rubredoxin at shorter wavelengths is not observed in the reduced forma of any of the other non-heme iron proteins examined. These distinctive
MCDsignals may prove characteristic
iron-sulfur
coordination.
more detailed
However, such conjectures remain to be explored in
investigations
now underway.
Systematic studies of synthetic properties
for high-spin tetrahedral
analoguea wilth structural
akin to those of the iron-sulfur
by Holm and his collaborators
proteins,
and electronic
such as reported recently
(20X and,of other complexes of iron with nitrogen
and oxygen liganda are needed to discern the precise basis of the spectra observed so far. REFERENCES 1. 2.
Ulmer, D. D. and Vallee, B. L., Biochemistry 2, 1335 (1963). Vallee, B. L. and Wacker, W. E. C., "The Proteins," H. Neurath, Ed., Vol. 5, Academic Press, New York (1970). 3. Ulmer, D. D. and Vallee, B. L., "Bioinorganic Chemistry," Adv. in Chem. Series 100, R. F. Gould, Ed., Am. Chem. Sot. Publ., p. 187 (1971). 4. Buckingham, A. D. and Stephens, P. J., Ann. Rev. Phya. Chem. l7, 399 (1966). 5. Schatz, P. N. and McCaffery, A. J., Quart. Rev. Chem. Sot. (London) 23, 552 (1969). 6. Kaden, T. A., Holmquiat, B. and Vallee, B. L., Biochem. Biophya. Rea. Consnun.46, 1654 (1972). 7. Ulmer, D. D., Federation Proc. m, 738 (1971). 8. Gray, H. B., "Bioinorganic Chemistry," Adv. in Chem. Series 100, R. F. Gould, Ed., Am. Chem. Sot. Publ., p. 365 (1971). 9. Bayer, E., Bather, A., Krauaa, P., Voelter, W., Barth, G., Bunnenberg, E. and Djeraaai, C., Eur. J. Biochem. 22, 580 (1971). 10. Ehrenberg, A. and Laurell, C. B., Acta Chem. gcand. 2, 68 (1955). 11. Feeney, R. E. and Komatsu, S. K., "Structure and Bonding," Vol. 1, p. 149, Springer-Verlag, Berlin (1966). 12. Aisen, P., Liebman, A. and Reich, 8. A., J. Biol. Chem. 241, 1666 (1966). 13. Gaber, B. P., Schllllnger, W. E., Koenig, S. H. and Aiaen, P., J. Biol. Chem. 245, 4251 (1970). 14. Price, E. M. and Gibson, J. F., J. Biol. Chem.247, 8031 (1972). 15. Poe, M., Phillips, W. D., Glickaon, J. D., McDonald, C. C. and San Pietro, A., Proc. Nat. Acad. Sci. U.S. 68, 68 (1971). 16. Poe, M., Phillips, W. D., McDonald, C. C. and Lovenberg, W., Proc. Nat. Acad. Sci. U.S. 65, 797 (1970). 17. Okamura, P., Klotz, I. M., Johnson, C. E., Winter, M. R. C. and Willisma, R. J. P., Blochemiatry 2, 1951 (1969). 18. Herriott, J. R., Sicker, L. C., Jensen, L. 8. and Lovenberg, W., J. Mol. Biol. 50, 391 (1970). 19. Phillips, W. D., Poe, M., Weiher, J. F., McDonald, C. C. and Lovenbarg, El., Nature 227, 574 (1970). 20. Her&wits, T., Averill, B. A., Helm, R. H., Ibira, J. A., Phillips, W.D. and Weiher, J. F., Proc. Nat. Acad. Sci. U.S. 69, 2437 (1972). 1061