Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IDENTIFICATION OF THE SIXTH LIGAND OF METHIONINE SULFOXIDE CYTOCHROME c Kathryn M. Ivanetich, Jean J. Bradshaw and
G. Victor Fazakerley
Department of Physiology and Medical Biochemistry, University of Cape Town Medical School, Observatory, C.P., and Department of Chemistry, University of Cape Town, Rondebosch, C.P., South Africa. Received June 28,1976 SUMMARY: Relevant portions of the proton n.m.r, spectra of ferri and ferro methionine sulfoxide cytochrome c are reported. At neutral pH, resonances attributable to the-methyl group of methionine sulfoxide-80 are located at -5.3 and -2.45 ppm relative to DSS for ferri and ferro methionine sulfoxide cytochrome c, respectively. No such resonances are observable in the ferric compound at pH 4.5 where the derivative is in the high spin form. It is concluded that methionine sulfoxide-80 provides the sixth ligand in methionine sulfoxide cytochrome c. The liganding is definitely via the sulfur atom in the ferro derivative, and is probably also via this atom in the ferric compound. Methionine sulfoxide cytochrome ~ is a derivative of horse heart cytochrome c in which the methionine residues at positions 65 and 80 are converted to methionine sulfoxide residues.
Methio-
nine sulfo~ide cytochrome c is unique in that it is the only active derivative of cytochrome c in which the sixth axial ligand, methionine-80,
is chemically modified
(i).
It has been proposed
that methionine sulfoxide-80 provides the sixth
(axial)
ligand
to the heme iron ion in methionine sulfoxide cytochrome c (i). The identity of the sixth ligand, however,
remains to be firmly
established. Proton magnetic resonance studies have been very successful in elucidating the nature of the sixth axial ligand in both oxidation states of cytochrome c and derivatives We have, therefore,
thereof (2-6).
attempted to identify the sixth ligand of
ferri and ferro methionine sulfoxide cytochrome c using this technique.
Cop))right © 1976 by Academic Press, Inc. All rights o] reproduction in any ]orm reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
M A T E R I A L S A N D METHODS: M e t h i o n i n e s u l f o x i d e c y t o c h r o m e c was p r e p a r e d and p u r i f i e d a c c o r d i n g to the m e t h o d of I v a n e t i c h et al. (I). The d e r i v a t i v e c o n t a i n e d less than 7% u n m o d i f i e d c y t o c h r o m e c by analysis of the 695 b a n d content. This analysis was conf i r m e d by o b s e r v i n g r e s o n a n c e s of the u n m o d i f i e d c o m p o u n d in the p r o t o n m a g n e t i c r e s o n a n c e s p e c t r a of m e t h i o n i n e s u l f o x i d e cytochrome c. C i r c u l a r d i c h r o i s m s p e c t r a w e r e p e r f o r m e d on a Jasco JA4OB r e c o r d i n g s p e c t r o p o l a r i m e t e r . Proton magnetic resonance spectra were obtained with a B r u k e r 90 MHz fourier t r a n s f o r m n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o p h o t o m e t e r using a iO m m probe. For initial experiments, 36 mg of c y t o c h r o m e c or m e t h i o n i n e s u l f o x i d e c y t o c h r o m e c (1.94 mM) w e r e d i s s o l v e d in 1.5 ml of D20. For final results, 300--mg of c y t o c h r o m e c or m e t h i o n i n e s u l f o x i d e c y t o c h r o m e c (16.1 mM) w e r e d i s s o l v e d in 1.5 ml of D20. pD was a d j u s t e d w i t h DCI or NaOD. C h e m i c a l shifts are r e p o r t e d r e l a t i v e to DSS. Samples w e r e r e d u c e d by the a d d i t i o n of a 3-fold excess of s o d i u m d i t h i o n i t e after b u b b l i n g w i t h o x y g e n - f r e e n i t r o g e n for i0 min. The tubes w e r e then s e a l e d under n i t r o g e n and s p e c t r a of the r e d u c e d compounds w e r e o b t a i n e d w i t h i n 30 min of reduction. There was no sign of o x i d a t i o n of the samples at the end of these experiments. RESULTS: (16.1 mM),
Proton
magnetic
pD 7.2 and
resonance
spectra
ferri m e t h i o n i n e
of f e r r i c y t o c h r o m e
sulfoxide
cytochrome
/ A A B
8
i.
ZO
3'0
2'0
I(Dppm
b 6
-I'0
-2'0
-3'0 p p m
P r o t o n m a g n e t i c r e s o n a n c e spectra of f e r r i c y t o c h r o m e c and ferri m e t h i o n i n e s u l f o x i d e c y t o c h r o m e c. Room temperature; p r o t e i n c o n c e n t r a t i o n , 16.2 mM. (A) ferri m e t h i o n i n e s u l f o x i d e c y t o c h r o m e c, pD 7.5; (B) ferri m e t h i o n i n e s u l f o x i d e c y t o c h r o m e c. pD 4.5; (C) ferri c y t o c h r o m e ~, pD 7.2.
434
c
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
(16.1 mM), pD 4.5 and 7.5 are presented in Figure i. resonances at +35.5, +32.8 and -24.2 p.p.m,
The
in the spectrum of
ferricytochrome c appear to be shifted in the spectrum of the ferri methionine sulfoxide ~ toward the peak representing the majority of unshifted protons of the protein plus the water peak. In the spectrum of the derivative at pD 7.5, analogous resonances appear at 23.3, 20.9 and -5.3, respectively, whereas
at pD 4.5,
all shifted resonances have collapsed to under the main protein/ water peak,
and are not observable.
The proton magnetic resonance spectra of ferro methionine sulfoxide cytochrome c at neutral pH is presented in Figure 2. The unsplit resonance assigned to the methyl group of methionine80 in ferrocytochrome c is located at -3.3 p.p.m, protein
(3,4) and appears to be shifted to -2.54 p.p.m, in the
20 2.
in the native
1'o
6
7Opp
Proton magnetic resonance spectrum of ferro methionine sulfoxide cytochrome c. Room temperature; protein concentration 16.2 mM, pD 7.2.
435
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
derivative.
Proton
magnetic
solutions
of ferri
(1.94 mM)
exhibited
identical
centrated
(16.1 mM)
samples.
Circular sulfoxide
cytochrome
centrations not
Table
and ferro m e t h i o n i n e
dichroism
are i d e n t i c a l
resonance
peaks
spectra
to p u b l i s h e d
up to 2mM,
sulfoxide
cytochrome above
c
for con-
and 2 mM ferri m e t h i o n i n e
sodium
spectra
of dilute
as d e s c r i b e d
of O.13
c in O . O 2 M
spectra
phosphate
(i) i n d i c a t i n g
the c o n f o r m a t i o n
buffer, that
pH 7.5,
at con-
of the d e r i v a t i v e
was
altered.
I.
S h i f t e d resonances of c y t o c h r o m e s u l f o x i d e c y t o c h r o m e c. a
pD
Compound
c and m e t h i o n i n e
Resonances Porphyrin Methyls
Unresolved Methyls
Fe +3 cyt c
7.5
Fe +3 m e t h i o n i n e s u l f o x i d e cyt
7.5
Fe +3 m e t h i o n i n e s u l f o x i d e cyt c
4.5
Fe +2 cyt c c
6.4
-0.7
Fe +2 m e t h i o n i n e s u l f o x i d e cyt c
7.2
-0.7
35.5,
33
(p.p.m.) Met (sul foxide) -80
-24.2
23.3,20.~ (35.5,33) u
-5.3 (-24.2) b
n.o.
n.°-)b
(35.5,33) b
(n.o
-3.3 -2.45 (-3.3) b
a
Abbreviations :
cyt c, c y t o c h r o m e
c_;
n.o.,
not observable.
b Values in p a r e n t h e s e s are r e s o n a n c e s arising from c y t o c h r o m e contamination. The intensities of these resonances are approxim a t e l y 7-10% of c o m p a r a b l e resonances of m e t h i o n i n e s u l f o x i d e c y t o c h r o m e c. C
From
reference
4.
436
Vol. 72, No. 2, 1976
DISCUSSION:
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cytochrome c has been extensively investigated
using the technique of proton magnetic resonance 2-6).
Because
(see for example,
of the electronic nature of the heme moiety, the
resonances of protons in close proximity to the heme group are drastically shifted from their normal positions.
Resonances
upfield and/or downfield from DSS are clearly dissociated from the compl~x, heavily overlapping region of the spectrum p.m.) associated with the normal protein resonances.
(ca. 0-iO p.
For ferri-
cytochrome c, markedly shifted resonances have been assigned unequivocally to the protons of the heme ring
(i.e.
+32.8,
+35.4 p.p.m.)
and those of the sixth axial ligand of cytochrome c,
methionine-80
(-24.2 p.p.m.)
(6).
We have taken advantage of the shift in the position of the resonances
associated with methionine-80 to investigate the nature
of the sixth ligand in a derivative of cytochrome c in which the normal sixth ligand, methionine-80 is chemically modified to methionine sulfoxide-80. characterized,
This derivative has been extensively
and the sixth ligand has been proposed to be
methionine sulfoxide-80 bound via its sulfur atom evidence
for this proposal, however,
is lacking.
(i).
Direct
The p r o t o n
magnetic resonance studies of methionine sulfoxide cytochrome c discussed below indicate that methionine sulfoxide-80 is coordinated to the central iron atom of ferri and ferro methionine sulfoxide cytochrome c. In the p.m.r,
spectra of ferri and ferrocytochrome c
prominent unsplit resonances a DSS standard.
appear shifted far upfield from
These resonances have been assigned to the un-
split methyl group of methionine
80, and their large shift
can only be explained as arising from their extremely close proximity to the heme group.
Less prominent, broadened re-
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BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
sonances assigned to the B and y methylene protons of methionine80, visible at high resolution, both ferri and
are also shifted greatly in
ferrocytochrome c
(2-4).
The high-field methyl
resonance of methionine-80 appears at -3.3 p.p.m, in ferro
and ferricytochrome c, respectively
and -24.2 p.p.m.
(3,6).
0
For ferro and ferri methionine sulfoxide cytochrome ~, analogous unsplit sharp signals occur at -2.5 p.p.m, p.p.m,,
respectively.
and -5.3
The unsplit sharp nature of these peaks
strongly suggests methyl resonances.
The large shift upfield of
these peaks is most likely explained by the close proximity or liganding of an atom adjacent to this group to the heme iron. In ferri methionine sulfoxide cytochrome c, the shifted resonance is equal in intensity to that of each of the two methyl resonances of the porphyrin ring, substantiating the assignment as a methyl group.
Of possible strong field ligands such as lysine, histidine
or methionine, resonance.
only methionine could give rise to an unsplit methyl
The resonances of the single 8 and y methylene protons
which could further support this proposal are not clearly discernible in ferrocytochrome c or methionine sulfoxide ferrocytochrome under the conditions of our experiments.
For methionine
sulfoxide ferricytochrome c, the large magnitude of the shift of peaks assigned as the methyl resonance of methionine sulfoxide80 ring
(-5.3 p.p.m.)
and the 2 methyl resonances of the porphyrin
(23.3 and 20.9 p.p.m.)
compared to ferricytochrome c may
be due to a drastic alteration in the environment of the heme moiety accompanying the coordination of methionine sulfoxide as the sixth ligand.
It has been reported that the heme
crevice of this derivative is weakened relative to cytochrome c ( 1
), and thus the environment of the heme crevice must be
altered compared to cytochrome c.
438
Similar shifts in the positions
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
of the resonances a s s i g n e d to the methyl groups of the porphyrin ring
( 22.9,
21.1) have been reported for cyanoferricytochrome
c m
(3).
In this derivative cyanide has displaced methionine-80
as the sixth ligand and the heme crevice is weakened relative to ferricytochrome c° The similar positions of the methyl resonances of methionine-80 in ferrocytochrome c (-3.3 p.p.m.)
and of methionine sulfoxide-80
in methionine sulfoxide ferrocytochrome
c (-2.45 p.p.m.)
indicate
that the liganding is via the sulfur atom of methionine sulfoxide-80 Were the liganding via the oxygen atom, a much smaller shift than observed might be expected in view of the greater distance between the methyl of methionine sulfoxide-80 and heme moiety.
It seems
likely that the liganding is also via the sulfur in methionine sulfoxide ferricytochrome c for reasons described earlier however,
(I);
our data does not unequivocally support this position.
Acknowledgements: This work was supported by grants from the S.A.M.R.C. and U.C.T. Staff Research Fund.
REFERENCES i.
Ivanetich, K.M., Bradshaw, J.J., Biochemistry, 15, i144-i153.
2.
Kowalsky, A.
(1964)
Biochemistry 4, 2382-2388.
3.
W~thrich,
(1969)
Proc.Natl. Acad. Sci. U.S. 63, 1071-1078.
4.
McDonald, C.C., Phillips, W.D., and Vinogradov, Biochem. Biophys. Res. Commun. 36, 442-449.
5.
W~thrich, K., Aviram, I., and Schejter, A. Biophys. Acta, 253, 98-103.
6.
McDonald, C.C. and Phillips, W.D. 3170-3186.
K.
439
and Kaminsky, L.S.
S.N.
(1971)
(1975)
(1969)
Biochim.
(1973) Biochemistry,
12,