Download Aron Janine 1986

THE
COORDINATION
AND
IRON
CHEMISTRY
PORPHYRIN
OF
SOME
COBALT
COMPLEXES
Janine Aron
A Dissertation submitted to the Faculty of Science,
University of the Witwatersrand, Johannesburg,
for the Degree of Master of Science
May 1986
C O R RI N O ID
ABSTRACT
The r6le of conformationa change in the protein's control of the
active metal site in haemoproteins and enzymes is examined with
(i) cobalt corrinoid cofacvors for the B -dependent isomerases,
(ii)
cytochrome
c,
and
(iii)
a model
for
cytochrome
c,
microperoxidase-8.
The half-lives (t/$) of homolytic fission of the cobalt carbon
bond ir the B
coenzyme, adenosylcobalamin, and in methyl, ethyl
and cyclobutylcobalamins have been determined in aqueous solution
at 96 °C. The t!£ values increase (rates decrease) in the following
ligand order:
ethyl
(ca.
9 min)
< cyclobutyl
(ca.
75 min)
< adeno^yl (c*». '80 min) < methy) (ca. 80 hrs). These values have
been used to provide an estimation of
ca. 10 years for the rate
in the protein-free coenzyme at room temperature. Comparison with
published data on the B -isomer^ses shows that the Co-C bond in
the coenzyme is lat^jlisea by ^ 10' when bound by the apoenzymo and
by a further ^ 10
in the p r e s e n c e of substrate to achieve the
overall labilisation of >,10
(i.e. t’
>} ■
v 2.5 ms) required for the
enzymatic reaction.
The
pK
for
the
alkaline
isomerisation
of
hor?*e-heart
cytochrome c (from Type III to Type IV, with cleavage of the
iron-methionine (Fe-S) bond) has been confirmed to be 9.05 at 25°C,
and extinction coefficients for Type III and Type IV at 695 nm are
867 and 83 M
cm
respectively. Binding studies with cytochrome
c in aqueous solution at 25 °C monitored the 695 nm charge-transfer
absorbance band, present when the Fe(III/-S bond is intact. The
specific binding of the ^perchlorate anion to cytochrome c occurs
with K (
ca. 5 x 10
M
(1:1 complex) and coulombic and
chaotropic effects are implicated. Donor-acceptor complexes of
caffeine
and tryptophol
with
cytochrome c
are
formed
where
^
is ca. 6 x 10
M
(1:1 complex) and K
is ca. 9 x 10~£ M(?*?2 complex), respectively. Other drugs, including chlorpro m a z i n e,
procaine and indole acetic acid, demonstrate thes»* »- * interactions
with cytochror c and induce Fe-S bond cleavage.
An impro
ithod for the preparation and purification of
microperoxida^
(MP-8) and a TLC method as a rapid test of
purity, have been developed. The binding of thioethers to MP-8
(possible models for the Fe-S bond in cytochrome c) ana of drugs to
MP-8 has been studied in 20* Me0H:H,0 (v/v> solution at 25 °C,
where 2 x 10
M
MP-8
is
> ^7% monomeric
at
pH
7.4.
N-acetyl-D, L-m*thionin«* binds to MP-8 with K
ca. 1.9 M~ at pH
7.4 and K
ca. 2.1 M
at pH 6.7 (1:1 compleSPs): the 695 nm band
characteristic of th" cytochrome c Fe-S bnnci is exhibited
D,L-methionine binds to MP-8 with K
ca. 155 M
at pH 7.4 (1:1
complex). Doth caffein*- and tryptojSRol form 1:1 donor-acceptor
complexes with MP-8: K
is ca. 151 M~ at pH 7.4 and ca. 240 M
at pH 6.7, for caffeinP? and K
is ca. 60 M
at pH 7.4, for
tryptophol.
Other
drug
molecfi?es,
including
chlorpromazine,
demonstrate these n - ti interactions with MP-8.
Donor-acceptor
complexes are formed with greater ease with these mode) complexes
than with cytochrome c.
(iii)
Declaration
I hereby declare that the work
carried out
exclusively
reported
by me and
that
in this dissertation
was
the dissertation has not
been submitted for a degree at any other university.
T & m r u .
,-lrov-L
Janine Aron
And a H**v«n }n «i Wild Flow#r,
Hold Infinity in th* p«]« of yoyr h»nd
And
in «n hour.
tftlllM
Blah*
ACKNOWLEDGEMENTS
I should like to expreaa my gratitude to the following:
Dr. D. Baldwin and Professor J.
Pratt,
my
supervisors,
for
their
guidance and encouragement;
Mrs. I. Aron, Mr. J. Unterhaltsr and Dr. B. Unterhaltcr for their
kindness, generosity and support;
Dr.
S.
Chemaly
and
Mr.
H.
Marques
for
helpful
discussion
and
advice;
The technical staff of the Department of Chemistr>, in particular:
Mr. J. Zimmermann,
Mr. S. van der Schyff,
Rev. a . Molefe,
Mr. A.
Thoane and Mr. B. Fairbrother;
Dr. P. Adams for permission to report the HPLC results concerning
microperoxidase-B prepared in this study;
Mr A. Domleo of Glaxo-Allenbury (S.A.) (Pty) Limited for samples of
vitamin
, and vitamin
Maybaker (S.A.) (Pty) Limited for the gift of chlorpr< ni»xine;
Mrs. L. Meredith for typing this dissertation;
Mrs. M. Crabb for technical assistance with diagrams;
Mrs. S. Dobson, Mr. B. Jersky, Ms.
assistance with proofreading.
J.
Mailer
and Mr.
G.
Yeo
for
(Vi)
TABLE OF CONTENTS
CHAPTER 1 - INTRODUCTION
1
1.1
Metallo-enzymes
1
1.2
Cobalt corrinoids used in this work
?
1.3
Iron porphyrins ured in this work
11
1.4
The aims of this dissertation
23
CHAPTER 2 - MATERIALS h NI) METHODS
24
2.1
Materials
24
2.2
Methods
26
CHAPTER 3 - THE PREPARATION AND PURIFICATION OK
ORGANOCORRINOIDS
3.1
3V
Features of preparation, purification and
identification
37
3.2
The preparation of organocobalamins
38
3.3
The preparation of oiganocob*namid*5
39
3.4
Summary
44
CHAPTER 4 - HOMOLYTIC THERMOLYSIS OF ETHYL, METHYL AND
CYCLOBDTYLCOBAl.AMINi; AND CORRESPONDING
COBINAM IDES, AND ADENOSYLCOBALAMIN
45
4.1
Introduction
45
4.2
i-Ellmtnation and homolytic fission
45
4.3
The rflle of steric strain in cobalt-carbon
bond cleavage
46
4.4
Photolysis of organoccbalamins
48
4.5
Adenosy lcoba 1amin-d»*pendent isomerase reactions
50
4.6
The
51
4.7
Experimental procedure
54
4.8
Results
55
4.9
Evidence for homolytic thermolysis
63
4.10
The
room
rflle of the protein in isomerase reactions
order
of
ligands
temperature
and
half-1 iff
decomposition of the coenzyme
estimation of the
for
homolytic
64
(vii )
TABLE OF CONTENTS (contd.)
Pace
4.11
4.12
Estimation
of
the
labilising
rfile
of the
protoin
64
Summary
66
CHAPTER 5 - STUD IKb >'?! THE REACTIVITY 0.T THE IRONKKTHIONINK
BOND
IN
CYTOCHROME
68
C
5.1
Introduction
68
5.2
Experimental procedure
72
5.3
The usp of unpurified commercial cytochrome c
without further
5.4
5.5
oxidation
74
The pK
of the alkaline isomerisation and
a
calculation of extinction coefficient* at 695 nm
75
The binding of small molecules and drugs to
cytochroaie c
81
5.6
Disruption
93
5.7
Summary
99
CHAPTER 6 - PREPARATION
OE
NICROPEROXIDASE-8
AND
ITS
ANALYSIS BY THIN-LAYER CHROMATOGRAPHY
101
6.1
Introduction
1C1
6.2
Experimental procedure
1C6
6.3
The preparation of mlcroperoxidase-8
lCd
6.4
The TLC system and R
108
6.5
values
“It
HPLC results for microperoxidase-8
112
6.6
Discussion
114
6.7
Summary
117
CHAPTER 7 - EQUILIBRIUM STUDIES WITH MICROPEROXIDASE-8 IN
AQUEOUS-MKTHANOL SOLUTION
118
7.1
Introduction
118
7.2
Experimental procedure
124
7.3
A monomeric system
125
7.4
Micror r o x idaso-8/thioetht'r complt*x«'s as models
for cytochrome c
7.5
7.6
127
Donor-acceptor interactions in microperoxidase8/drug complexes
133
Summary
1 37
(v i ii )
TABLE OF CONTENTS (contd.)
Pa8 e
CHAPTER 8 - SUMMARY AND CONCLUSIONS
APPENDIX
138
- Derivation of equations for kinetic studies
(Chapter 4) and equilibrium studies (Chapters
5 and 7)
REFERENCES
141
146
(ix)
L IST OF TABLES
Page
Table 1 ,1
E:>zyme reactiona requiring cobalt corrinoids
Table 3 .1
UV-visible
absorption
spectra
for
10
organo-
corrinoids in this work
Table 3 ,2
values
of
some
43
corrinoid
complexes
in
Solvent I
Table 4 ,1
The
44
half-lives
of
decomposition
organocorrinoid complexes
oxygen,
at
pH
6
and
7
under
of
some
nitrogen
and
(p H - i n d e p e n d e n t ),
at
96°C
Table 5 ,1
62
Recent
determinations
of
the
alkaline isomerisation and
and IV of cytoc
Table 5 .2
Equilibrium
perchlorate
pK
for the
a
for Types III
one c
constants
79
for
to cytochrome c
the
binding of
-4
(ca. 1 x 10
M)
in alkaline solution
Table 5 ,3
Equilibrium
constants
86
for
the
binding
of
caffeine and tryptophoJ. to cytochrome c (ca.
•4
1 x 10
M) in alk* 'ne solution
Table 6 1
HPLC
analysis of C‘ 'errial MP-8 samples %ni
those prepared as „*-•
Table 7 1
88
Equilibrium
various
constants
ligands
to
2 x 10"' M) in 20* fneOW
.bed in the text
for
the
monomeric
binding
MP-8
,0 (v/v) solution
1*4
of
(ca.
133
(x)
LIST OF FIGURES
Page
Fig. 1.1
Haemin
chloride
(Fo(III)
protoporphyrin
IX
3
chloride)
Fig. 1.2
The molecular structure of
Fig. 1.3
(i)
4
,
The structure of the cob^lamin coenzyme
(ii) The 5'-deoxyadenosy1 ligand present in
coenzyme
6
Fig. 1.4
The structure of cobinamides
7
Fig. 1.5
Organocorrinoid reactions
9
Fig. 1.6
Prosthetic groups of the cytochromes
Fig. 1.7.
Cytochrome c in
Fig. 1.8
The structure of cytochrome c
14
Fig. 1.9
The structure of microperoxidase-8
20
Fig. 3.1
Absorption
1?
muitienzyme system
spectra
of
Bj ,
aquocyanocobinamide ( —
Fig. 4.1
12
( —— )
and
) in wa*er
41
Scheme of possible reaction mechanisms for the
isomerase enzymes
Fig. 4.2
Scheme
of
50
reactions
initiated
by
homolytic
fission of th** Co-C bond in or&anoccrrinolds; R
may
be
methyl,
ethyl
or
ad«-ro*;.'l,
and
Co-R
denotes five - and/or six- coordit.ate species
Fig. 4.3
Spectral
changes
deccripos i t ion
of
under nitrogen:
obser
d
taer.osyl
during
r>al«tnn
at
53
the
9*. °C
A ■ 2 mlnu.;'«, B •> 66 minutes,
C ■ 145 minutes and D ■ 34* -.M.-nites
FI*. 4.4
First-order kinetic plots
56
.
►
*< decomposition
at 96 °C of a) a d e n o s y l c o t ' '-
>> under nitrogen
and b) e*hylcobalamln und« » 'hy***n
Fig. 4.5
Spectral
changes
obs
decomposition of e thy 1
t
56
during
the
1amir. at 96 °C under
nitrogen: A ■ 1 minute,
K ■
minutes, C *1 18
minutes «»nd D • 37 minutes
Fig. 4.6
Spectral
changes
58
obf»**rved
during
decomposition of ethyIcoblnamlde at 96
7,
under
oxygen:
A
-
3
minutes,
the
#C,
B
pH
■ 46
minutes, C ■ 264 minutes and D ■ 1598 minutes
58
LIST OF FIGURES (contd.)
Fig. 4.7
First-order kinetic
plot of
the decomposition
at 96 °C of etnylcobinamide under oxygen
Fig. 4.8
First-order kinetic plot
of
the decomposition
at 96 °C of methylcobnlamin under oxygen
Fig. 4.9
First-order kinetic plot of
at 96 #C
Fig. 5.1
the decomposition
of cyclobutylcobalamin under nitrogen
Relationship of haem to the polypeptide chain,
to the bottom of its crevice and to the surface
of the molecule
Fig. 5.2
The correction method of Kaminsky et a l . (1973)
for background absorbance
fig. 5.3
The
effect
of
purification
and
oxidisation
(with K^FefCN)^) of cytochrome c on the binding
of sodium p«.rchl rate: V purified cytochr me c;
• unpurified
cytochrome
c; o purified
and
oxidised cytochrome c
Fig. 5.4
Acid-base
titration of the 695 nm absorption
-4
band of 1.13 x 10
M cytocorome c at 25 #C:
(a) Af/)S against pH;
(b) A^iJt (corr.)
against
pH (u - 0.05)
Fig. 5.5
The data
Fig. 5.4 (a)
are plotted according
to equation 10 (Section 5.4.]
Fig. 5.6
Spectra of a titration of the 695 nm absorption
..4
band of ca. 1 x 10
M cytochrome c by sodium
p e r c h l o r a t w , pH 9.0: A « 0 M NnC<0.;
Fig. 5.7
B • 0.014
M NaC<0.; C - 0.053 M NaC«0„;
D • 0.14h M
4
4
NaCIO.
4
The corrected absorbance values at 695 nm as a
function of sodium porchloratr
concentration:
A ■ pH 8.8, B ■ pH 9.0, C ■ pH 9.3 and D ■ pH
-4
9.5; cytochrome c concentration
ca. 1 x 10
M (tris-hydrofhloric acid buffers, p ■ 0.1)
Fig. 5.8
The
binding of sodium perchlorate
to
ca.
-4
1 x 10
M cytochrome c: A - pH 8.8, B • pH
9.0,
C ■ pH 9.3
and
D • pH 9.5
chloric acid buffers, u ■ 0.1)
(tris-hydro-
(xii )
LIST
t FIGURES (contd. )
Pa*e
Fig.
.9
The
plots
of
«og
*^app
against
pH
for
perchlorate, caffeine and tryptophol binding to
>4
c*. 1 x 10
M cytochrome c. The vhJuo ol <?og
K
is given by the ordinate intercept
85
-4
Fig.
.10
The
695
nm
absorbance
cytochrome c
as
a
of
"a.
1
of
the
finctlon
x
10
M
increased
86
concentration of sodium nitrate
-4
Fig.
.1.
The
binding
cytochrome
of
c:
caffeine
to
A • 8.8,
ca.
1
B ■ 9.3
x
10
and
M
C • 9.5
(tris-hydrochlorlc acid buffers, u ■ 0.1)
89
-4
Fig.
.12
The binding of
tryptophol
to ca.
1 x
10
M
cytochrome c: A • pH 9.0, B ■ pH 9.3 and C ■ pH
9.5
Fig.
.13
(tris-hydrochloric acid buffers,
u » 0.1)
Representation of the electric potential
generated
within
human
from
the
left
cross-section
of
the
which
field
ferricytochrome
(source: Osneroff et a l . (1980)).
protein
lies
c
The view
looking
approximately
89
is
at
a
along
tne dipole axis and contains both the top haem
crevice salt bridge
between
of
the
lysine
13
and
the
c-amino group
a-carboxyl
group
glutamic acid 90 and the bottom hydrogen
of
bond
between the c-amino group of lycine 79 and the
backbone carbonyl of residue 47. The Italicized
numbers
Fig.
.1
denote
the
strength
of
the
electric
potential lines, in kTD |eD
w
p
The amino acid sequences of
the undecapeptlJe
of
and
cytochrome
c
(MP-11),
96
its
tryptic
product,the octapeptide (MP-H)
Fig.
.2
Preparation
of
MP-8:
column
103
chromatography
elution profiles. Solvent:
0.1 M NH.HC0*, room
4
3
temperature. Pooled fractions are indicated by
arrows.
(i) Peptic
digestion
product
of
cytochrome c on 4 x 100 cm Siogel P6 at 20
-2 -1
m<cm hr , 2.00 m€ fractions: A ■undigested
cytochrome
c,
B
■ MP-11,
C-E »
smaller
(xii)
LIST OF FIGURES (contd.)
Page
Fig. 5.9
The
plots
tog
of
^app
ag a inBt
pH
f°r
perchlorate, caffeine and tryptophol binding to
-4
ca. 1 x 10
M cytochrome c. The value of €og
is given by the ordinate intercept
Fig. 5.10
T.ie
695
nm
absorbance
cytochrome c
as
a
of
ca.
1
of
the
function
85
x
10 1
M
increased
86
concentration of sodium nitrate
-4
Fig. 5.11
The
binding
cytochrome
of
c:
caffeine
to
A « 8.8,
ca.
1
B * 9.3
x
10
and
M
C ■ 9.5
(tris-hydrochloric acid buffers, u * 0.1)
89
-4
Fig. 5.12
The binding of
tryptophol
to ca.
1 x
10
M
cytochrome c: A « pH 9.0, B » pH 9.3 and C * pH
9.5
Fig. 5.13
(t n s - h y d r o c h l o r i c acid buffers,
u * 0.1)
Representation of the electric potential
generated
within
human
from
the
left
cross-section
of
the
which
field
ferricytochrome
(source: Osheroff et al . (1980)).
protein
lies
c
The view
looking
approximately
89
at
is
a
along
the dipole axis and contains both the top haem
crevice salt bridge between
of
lysine
13
and
the
the
f-amino group
a-carboxyl
group
glutamic acid 90 and the bottom hydrogen
if
bond
between the c-amino group of lysine 79 and the
backbone carbonyl of residue 47. The <talicized
numbers
Fig. 6.1
denote
the
strength
of
the
electric
potential lines, in kTD |eD
w
p
The amino acid sequences of
the undecapeptide
of
and
cytochrome
r
96
(MP-11),
its
tryptic
product, th»* octapeptide (MP-8)
Fig. 6.2
Preparation
of
MP-8:
column
elution profiles. Solvent:
temperature.
arrows.
103
chromatography
O.i M NHjHCO^, room
Pooled fractions arc indicated by
(i) Peptic
digestion
product
of
cytochrome c on 4 x 100 cm Biogel P6 at 20
-2
-1
m€cm hr , 2.00 m€ fractions: A = undigested
cytochrome
c,
B
=
MP-11,
C-E
=
smaller
(xiii)
LIoT OF FIGURES (contd.)
Pe.ge
unidentified
haem-pe p t i d e s .
(ii)
Tryptic
digestion product of MP-11, as in (i) but
A
with
*
B =■MP-8, C ■ unidentified haem-o e p t i d e . (iii)
Purification of MP-8 on 1.5 x 30 cm Sepnadex
- 2 -1
G-50
Superfine,
4
m€cm hr ,
2.00
m<
fractions:
A,
B
* MP-11
and
an
unidentified
impurity; C * MP-8
Fig. 6.3
109
The separation of a mixture of haem-peptiries on
silica
gel
water/88
0.144 g
TLC
mt
in
the
rolvent
sec-butai.ol/0.2
KCN:
A
»
system
m<
0.88 M
unidentified
impurity found in commercial
88
m<
NH,/
haem-peptide
MP-8;
B * MP-8;
C * MP-11; D • cytochrome c
Fig. 6.4
HPLC analysis of MP-8 on
temperature.
25 - 5C
sample gradient
2.25,
B
=
increased
Vydac
A
* TEAP
acetoritrile
linearly
C-18
at
injection of
eluted;
60/40
111
1 mg/m<
buffer,
in
to 60% B over
room
A:
20
10%
min
pH
B
and
then to 95% B over 0.5 min.
Detection at 395
nm:
purification
a)
This
work,
final
on
Sephadex G-50; b) Commercial MP-8 (Sigma)
113
Fig. 7.1
The structure of microperoxidase-8
119
Fig. 7.2
Beer's Law plot for MP-8 at 25 °C, u * 0.1, pH
7.4 (phosphate) in 1 cm and 10 cm cells in 20%
MeOH:
H^O
(v/v).
The
ordinate
values
ore
normalised for 1 cm cells
Fig. 7.3
128
The binding of N-acetyl-D,L-methionine
to ca.
2 x 10"'' M MP-8 in 20% Me0H:H20 (v/v) solution
at 25 °C in « 0.1 (phosphate)):
A ■ pH 6.7;
B
* pH 7.4
131
-5
Fig. 7 •4
Absorption spectra of ca.
1.6
M
Soret
N-acetylregion
D ,L-meth i o n i n e , pH
A:
methionine adduct;
A:
cytochrome
adduct; C: MP-8
2 x 10
c;
MP-8;
(ii)
B:
near
:
M MP-8
7.0:
in
(i)
MP-8/N-acety1infra-red
region
MP-8/N-acetyl-methionine
1 31
(x i v )
LIST OF FIGURFS (contd).
Fig. 7.5
Spectral
changes
2
x 10‘6
for
M
a
titration
MP-8
in
20%
of
ca.
MeOH:
H ,0
solution, pH 7.4 at 25 °C, with D ,L-methionine
Fig. 7.6
The binding ot D ,L-methionine to ca. 2 x 10
(v/v)
132
M
MP-8 in 20% Me0H:H,0 (v/v) solution at 25 °C (u
■ 0.1 (phosphate)) at pH 7.4
Fig. 7.7
Spectral
charges
2
for
a
132
titration
of
ca.
x 10~h M MP-8 in 20% Me0H:H,0 (v/v) solution,
pH 7.4 at 25 #C, with caffeine
Fig. 7.8
135
The binding of caffeine to ca. 2 x I0“6 M MP-8
in 20% M.;0H:H?0 (v/v) solution at 2 5 8C (u « 0.1
(phosphate)) at pH 7.4
Fig. 7.9
The binding of
MP-8
in
20%
135
tryptophol
M e O H :H ,0
to ca.
(v/v)
2 x
solution
(u ■ 0.1 (phosphate)) at pH 7.4
10~6
at
M
25°C
136
CHAPTER 1 - INTRODUCTION
1.1
M e t a llo-enzywes
Enzymes
are biological
catalysts
found
throughout
the
plant
animal kingdoms which catalyse a diversity of reactions
and
essential
to life. The metabolic rates of hundreds of pathways ire controlled
by enz/mes;
and
they usually
steric,
for
one
display high
or
a
substrates. Metallo-enzymes
small
specificity,
number
complex
cobalt
with
one
corrinoids)
cloaely
form a sub-group of
represent a partnernhip between a metal
metal
of
or
more
providing
the enzymes,
(e.g.
active
iron
or complex ( ‘
prosthetic group',
the
enzyme.
metabolism
Metallo-enzymes
cata
*ite,
such
as
the
or
important
enzymatic
and
a
metal. The
together w.
'co-factor*
are
and
porphyrins,
protein which controls and enhances the activity
protein is termed the 'apoenzyme* and,
related
ion (e.g. copper(II)) or a
ligands
the
both chemical
ie rr,e*al ion
'coenzyme'),
in
fixation
many
of
forms
areas
of
nitrogen
(by
nitrogenase enzyme*), energy production or interconversion (e.g. by
cytochromes)
ana detoxification mechanisms
in
the
body
(e.g.
by
p**roxliases, catalases and cytochrome P-450). Some of the enzymatic
reactions do not have analogues among protein-free complexes,
as carbon
(tkMeton
isomerisation
b/ the
isomeras** enzymes
such
(which
incorporate cobalt corrlnoid co-factors).
The enhanced catalytic activity of metallo-^nzymes Is due to a
modification
by
the
protein. The prosthetic group is tightly bound by the protein
the
binding
of
constant
the
can
properties
be
as
transferrin (Raymond et al.,
ligands
nd stereocnemistry
of
the
high
a
tetrahedral
rather
in
1982)). The valency of the metal,
the
fixf»d
protein,
usually
by
than
Bterlc
10*1 (e.g.
by
the
protein,
(e.g. copper(II)
conf igurntior., In p l a s t o c y a n i n ). Some
the
its
usual
means,
while
sometimes
is forced to
square
pathways may
introduced by the suitable positioning of groups
The protein controls
group
iron(III)
are
as
inducing strain in the metal complex
adopt
prosthetic
planar
be blocked
by
others
may
such as
thiols.
and modifies possible pathways
to
give
be
the
desired rate enhancement and specificity by varying one or more of:
(a)
the kinetics of
individual
steps,
(b)
the
thermodynamics
of
individual steps or (c) the thermodynamics of the overall reaction.
A variation of
thermodynamic properties
protein conformation,
catalytic
activity
coupled
is the predominant
mode
metallo-enzymes
(Pratt,
of
with
for
changes
in
enhancing
1975).
the
Thus
the
protein may
(i)
alter
the
equilibrium
particular
constant
for
the
ligand by electronic or
coordination
steric
means
of
a
(e.g.
the
in
the
coordination of oxygen by irun(IT ) in haemoglobin);
(ii)
stabilise
s
ligand
protein-free
coordination
species
five-coordinate
by
number
steric
mono-imidazole
unusual
means
iron(II)
(e.g.
the
porphyrin
in
peroxidases, catalases and cytochromes);
(ili) vary
the
changing
redox
the
potential
of
hydrophobicity
the
of
metal,
its
for
example
environment
(e.g.
by
in
cytochrome c);
(iv)
couple changes in the thermodynamic properties cf the metal
Site with the binding of the substrate (e.g. induction of the
normally unfavourable homolytic fission of the cobalt-carbon
bond in the cobalt corrinoid cofactors of isomerase enzymes);
(v)
couple changes
involving sites
distant
from
the metal
with
equilibria involving the metal (e.g. in haemoglobin).
For
a
more
extensive
review
of
the
r81e
of
iron protoporphyrin
IX
the
protein
in
metallo-enzymes, see Pratt (1975).
The prosthetic group
provides an excellent example
prosthetic group are altered
different
proteins.
protoporphyrin
TX
to show how
display
the propert
in consequence of
Different
great
(see Figure
haemoproteins
versatility
the at
s of
the
ichment of
containing
and
1.1)
iron
diversity
of
function. Thus, haemoglobin and myoglobin are respiratory carriers
which reversibly bind oxygen for transport o
Brunori,
1971);
the
cytochromes
have
electron transport in the mitochondrial
and
Barrett,
hydroxylate
1973);
mono-oxygenases,
hydrocarbons
(Hayaishi, 1962);
through
as
storage (Antonint and
principal
function,
respiratory chain (Lemberg
such
the
as
cytochrome
activation
of
P-450,
oxygen
catalases catalyse the decomposition of hydrogen
3
c h 2*c h
CH]
CHjCM^OOH
Fig. 1.1
CH jC H jCOOH
Haemin chloride (Fe(III) protoporphyrin IX chloride).
peroxide, wnile peroxidases use hydrogen peroxide to oxidise a wide
range of substances
(catalyses,
substrates of peroxidases, may
different
1984);
specificity
(Dixon
terminal
oxidases
and
which do not oxidise most of
be
regarded
and
Webb,
like
as peroxidanes
1971))
cytochrome
(Frew
c
the
with
a
and
Jones,
oxidase,
reduce
oxygen to water (Caughey et a l ., 1976).
In this dissertation,
metallo-enzymes
has
corrinoid-dependent
corrinoid models,
the rflle of the protein in two types of
been
studied:
isomerase
and
(ii)
enzymes
(I)
using
the haemoprotein
the
cobalt
protein-free
cytochrome c,
cobalt
and
a
model for cytochrome c, the haem-octapeptide, microperoxidnse-8.
1.2
Cobit It corrinoi<H uned in this work
(a ) History
Vitamin
B 1 , is
derivative
not
of
synthesized
>y
the
adenosylcobalamin,
cobalt-cofactor
for
methyl-malonic
and
methylmalony1-coenzyme
the
in
succinic
A
mutase
vivo
acids
(the
only
mammals requiring this B ( , coenzyme) e.g.,
0
i'.uman
is
body.
the
Yet
essential
interconversion
by
known
a
the
of
enzyme
reaction
in
A deficiency of vitamin
causes
pernicious
, in the diet, or its maladsorption,
anaemia.
Minot
and
Murphy
(1926)
first
reported the alleviation of this condition by feeding patients
raw liver; two decades later vitamin B ^ ^ (c y a n o c o b a l a m i n ) (see
Figure 1.2) was isolated by Folkers and co-workers
(Rickes
et
a l ., 1948) in the same year as Smith and Parker (1948). Vitamin
Bj j is today prepared by bacterial
fermentation rather than by
isolation from liver.
The crystallographic work of Hodgkin (Hodgkin et al., 1956)
aided by the chemical studies of Todd end Johnson (see Bonnet
et
al.,
1957)
established
that
B^ ,
contains
5,6-dimethylben2imidazole and cyanide anion as fifth and sixth
ligands
to
a
cobalt(III)
ion.
The
cobalt
ion
resides
corrin ring (which provides four planar nitrogen
to the porphyrin ring
in haemoproteins
(see
in
donors)
a
akin
tne structure
in
Figure 1.2).
A
biologically
coenzyme)
was
discovered
crystallographic
established
. 1.2
active
that
studies
the
form
by
of
of
vitamin
Barker
Lenhert
macrocyclic
et
B^?
(vitamin
B,
al.
(1958).
The
and
structure
Hodgkin
and
(1961)
peripheral
The molecular structure of B ( , (source: Pratt (1975)).
A deficiency of vitamin
causes
pernicious
, in the diet, or its maladsorption,
anaemia.
Minot
and
Murphy
(1926)
first
reported the alleviation of this condition by feeding patients
raw liver; two decades later vitamin
(c y a n o c o b a l a m i n ) (see
Figure 1.2) was isolated by Folkers and co-workers
(Rickes
et
a l ., 1948) in the same year as Smith and Parker 11948). Vitamin
Bj ^
today prepared by bacterial
fermentation rather than by
isolation from liver.
The crystallographic work of Hodgkin (Hodgkin et a l ., 1956)
aided by the chemical studies of Todd and Johnson (see Bonnet
et
al.,
1957)
established
that
B^.,
contains
5,6-dimethyIberizinidazole and cyanide anion as fifth and si..ih
ligands
to
a
cobalt)III)
ion.
The
cobalt
ion
resides
in
corrin ring (wh.4 'h provides fcur -lanar nitrogen donors)
to the porphyrin ring
>n hacmoproteins
(see
a
akin
the structure
In
Figure 1.2).
A
biologically
coenzyme)
was
diacovered
cryatallographic
established
g. 1.2
active
that
studies
the
form
by
of
of
vitamin
Barker
Lenhert
macrocyclic
The molecular structure of
et
»l.
and
structure
(vi‘*min
B, ,
(1959).
The
Hodgkin
and
(1961)
peripheral
, (source: Pratt (1975)).
substituents
of
the
coenayme
were
w hs
cyanocohalamin, but that cyanide
the
same
as
in
replaced by the 5' carbon
of an adenosyl group. The resulting Co-C sigma bond represents
a
unique
feature
.erivatives
of
of
metals
these
corrinoids:
sigma-bonded
alkyl
of the first transition series were at
the time of the discovery of
rather unstab!*?. Coenzyme
the
ccenzyme
considered
, (adenosylcobalamin)
to
be
(see Figure
1.3) was the first naturally occurring organometal1ic compound
to
be
discovered,
and
adenosylcobalamin
alkylcobeiamina remain the
nly known
enzyme!, wat
cofartcr
Ro s f
was
recently
be
• lologically
metabolism
(as
homocysteine
later discovered
in vivo.
determined
related
orginometal1ic complexes
in nature- Methylcobalamin, known to
and
essential
for
human
5
N -metrvltetrahydrofolate
and
active
cofactor
for
methyltransferase
The structure of
by
X-ray
this
crystallograpny
et a l ., 1085).
I
l?"^, the total synthesis of vitamin
, was reported by
WondwrtT'i (Woodward, 19"79>.
g true-are
am
-Jbalamin, adenosylcooalamin and methylcobalamin form part
a family of compounds
known as corrinoids.
The
naturally
o rrurring corrinoid all possess the same conjugated corrin ring
«»
B
,
incorporating
Corrinoids may differ
axial ligands;
itself and
a
central
cobalt(III)
in the nature
of
the
met*l
side
ion.
chains
and
rhoee that possess the same side chains as B ^
the nitrogen atom of 5,6-dimethylbenzimidfazole
fifth axial
ligand, are called cobalamins.
propionic
acid
attached
(R)-l-amino-2-propanol , and
to
C^7
the other
In ccbalamins,
is
amidated
side-chains
as
the
with
at Cp,
C <t
C 7 , Cg, C J3 and C^fl terminate in carboxyl groups, converted to
amide groups.
almost
All the propionamide groups point away from
planar
acetamide
macrocycle
side-chaina
in
in
the
one
direction,
other
and
direction.
all
The
the
the
other
nitrogen of 5,6-dimethylbenzimidazole is bound to ribose in an
ot-glycosidic linkage and th»- ribose moiety is bound,
via
a
phosphate
Cobinamides
are
linkage,
to
the
aminopropanol
in turn,
side-chain.
corrinoids where the phosphate linkage to the
6
<>
^CMOM
< . _~CmOm
&
*?CH,
r
(ii)
Fig. 1.3
(i)
The
structure
(source:
(ii)
The
moiety
of
The
replaced
sixth
the
benzimif* -ole
position
ligand
present
coordinated
ligand,
in
side-chain
in
often
vitamin
has
been
benzimidazole
cyanide
B ( , t3
cyanide (CN~). Cyunocobalamiri is a diamagnetic,
complex containing the d
coenzyme
(source: Pratt (1975)).
by another
axial
cobalamtn
5'-deoxyadenosy1
hydrolysed (see Figure 1.4). The
usually
the
Lenhert and Hodgkin (1961)).
coenzyme
ribose
of
or
is
water.
occupied
by
six-coordinato
cobalt(III) ion. The positive charges
on the cobalt ion are offset by the single negative charges on
the
corrin
ring,
the
cyanide
and
the
phosphate.
Adenosylcobalamin
(Figure
1.3),
methylcobalamin
and
other
cobalamins are also diamagnetic and may formally be regarded as
complexes of cobalt(lll) with a carbanion
(Abeles and Dolphin,
1976).
Reduction
of
cobalt(lll)
corrinoide
with
thiol
under
anaerobic conditions results in cobalt(II) complexes (Jaselskis
7
and
Diehl, 1954;
Dolphin et al., 1964)
which are low spin d
Co
CH,-
I N > ^
HOCH
. 1.4
The structure of cobinamldon:
hydrolysis at — * of
Cj7
gives
uidechain
in
cobalamins
the
the
corresponding
cobinamide.
compounds with one unpaired electron.
cobalt(II)
complex
is
probably
In neutral
solution,
five-coordinate
with
benzimidazole base coordinated to the cobalt
(Saveant,
Further reduction of
or
cobalt(III)
with
the cobalt(II)
sodium
borohydride,
which has been e^tablijhed
(Hill
et
aqueous
al.,
196?).
solution
nt
complex,
to be
gives
pH
to
the
1979).
reduct-i^n of
a
green
complex
a monovalent
cobalt
complex
C o ( I ) corrinoids
low
the
give
rapidly
Co(II)
decompose
and
in
hydrogen,
piobably
Co(II)
via an
intermediate cobalt
hydride.
Both
Co(I)
complexes are easily oxidised by t»ir to give
and
Co(III)
Q
complexes. C o (I > c^rrinoids are spin paired d
complexes.
The
benzimidazole
cobalt
the
base
is
not
coordinated
to
the
in
cobalt (I) B ] > complex (Brodie and Poe, 1971).
The
B,_
(cyanocob a l a m i n ),
B,_
12
12a
(c o b ( 1 1 )a l a m i n ) will be used in this
abbreviations
(aquocobalamin), and B , .
1 <?r
dissertation.
) Organocorrinolds
Both adenosyl
and methylc''balamins
prepared from B [ , or Bj
alkylcobalarrins
f: -m ethyl
'
1).
All
and
have
been
as have many primary and secondary
cobinamides,
and vinyl
coenzymes)
to
with
1-norbornyl
organocorrinolds
alkyl
ligands
(Schrauzer
possess
a
ranging
and
Holland,
cobalt-carbon
(Co-C)
sigma bond which cleaves homolytically in the presence of light
to
give
cubalt(II)
and
an
organic
free
radical.
ader.osylcobalamin and methylcobalamin are extremely
However,
stable
in
the dark both in the solid state and solution (see Chapter 4);
this applies to most o r ganocorrinoids, unless the organoligand
is particularly sterically hindered (e.g. isop^opylcobalamin).
The usual
mode of preparation of organocorrinoids
the reduction of a cobalt(III)
or
hydroxocc-balamin)
oe
cobalamin
(e.g.
cobalt* III)
corrinoid
(termed
cyanocobalamin
cobinamide
aquocyanocobinamlde or d i a q u o c obinamide) to
cobalt(I)
is via
(e.g.
the corresponding
"supernuclecphile"
by
Grate
and
Schrauzer (1979)), which is then reacted with compounds such as
alkyl
halides,
expoxides,
alkenes
or
alkynes,
to
give
the
desired product via S^2 substitution (see Chapter 3) e.g.,
NaBH
Co( 1 1 1 )-0H , ---- =*
Or^nccorrinoida
R-Ha<
C o d ) ------>
engage
in
a
diversity
involving th* cobnlt 'oordination sphere;
in Figure 1.5.
Co( III )-R
of
reactions
rhese are illustrated
Co-R
Co( I I )«-R‘
- HOMOLYTIC FISSION
Co-R
C o ( I )+R*
- REDUCTION
Co-R
Co-H-t-R-H
- 6 -ELIMINATION
Co-R
Co(IIIWR“
- HETEROLYTIC FTSSION
Co-R
Co-R
- 0RGAN0L1GAND MODIFICATION
Fig. 1.5
Organocorrinoid reactions.
(d; Stable yellow corrinoids
Stable yellow corrinoids are sometimes formed as by-products of
reactions with cobalamins that involve a change in the cobalt
oxidation state. They are thought to arise from a fr*tff radical
attack
on
the
corrin
ring:
see
for
example,
Grlining
and
Gossauer (1979). Stable yellow corrinoids (SYC) display a band
at about 460nm in their absorption spectra; they are impervious
to the effect of light, oxygen or cyanide, unlike
(e ) Enzyme reactions requiring corrinoids as cofactors
Three types of reactions in living organisms are catalysed by
enzymes containing cobalt corrinoids as c o f a c t o r s 1
1.
isomerisation
reactions
involving
the
1,2-shift
of
a
carbon, nitrogen or oxygen atom;
2.
methyl transfer reactions, for example in the formation of
methionine;
3.
the
conversion
of
ribonucleotides
to
2'-deoxyribo-
nucleotides, by the enzyme ribonucleotide reductase.
The
enzyme
reactions
adenosylcobal. min
requiring
and
(ii)
cobalt
corrinoid
methylcobalamin
(in
cofactors
methyl
(i)
transfer
reactions), are classified and tabulated in Table 1.1.
The
.,-dependent
isomerases
together
with
the
related
ribonucleotide reductases are the first aroup of metalloenzymes (as
opposed
to
reasonably
ha e m o p r o t e i n s )
well
understood.
where
The
the
rfile
mechanism
of
the
protein
is
whereby
changes
in
protein conformation are coupled to cnanges on the metal ion is not
known
for
certain,
but
several
mechanistic
proposals
have
been
10
reported (e.g. Pratt, 1984; Finke and Hay, 1984).
In this work only the
isomerase
reactions are considered.
In
Chapter 4, mode) studies with protein-free cobalt rorrinoids aim to
clarify the r51e of the protein in i&omerase snzymes.
For a recent
review of model studies concerning isomerase enzymes see Finke
et
al. (1984).
TABLE 1.1
Enzyme
reactions
requiring cobalt corrinoids
Chemaly (1980)).
Clot* 1 - Raoction* requiring oatncty icobolo*»in
L ____mat;--, ’
tgctior,,
Corbo«-»*•Itto"
Clutoi*ot* « u t o » *
thyl«alony l-co*n*yi««
A mutot*
1
Hijratio" of o"
omtne
H
1*2
D-
Co«v#riio« of
d i o l * or amino
a l c o h o l * to
aid*hyd«i
h
'
rt^v^ltondti
». ^ 2 ,- d t o « r i b o " v j c l » o ,.i,<'n
Bt3C t10"
OM OH
CM
♦(r s )2 ♦«2o
(R5H )2 . dithiol
1
B«aetior,
5 - C H j -THF ♦ H S ( C H 2 )2 C H ( N H 2 ) C OO H ------ ^
THF + CH3S(CH2;2CH(NH;)COOH
(source:
10
reported (e.g. Pratt, 1984; Finke and Hay, 1984).
In this work only the
isomerase
reactions are considered.
In
Chapter 4, model studies with protein-free cobalt corrinoids aim to
clarify the r81e of the protein in isomerase enzymes.
For a recent
review of model studies concerning isomerase enzymes see Finke
et
al. (1984;.
TABLE 1.1
Enzyme
reactions
requiring cobalt corrinoids
(source:
Chemaly (1980)).
Clot*
1
* Reoctiont
requiring
— ------ ^ e o r r ^ g e - e - t
or
oce«ctyicobaio»in
I to-- r
° »cc t I 0 " t
T«pe of reoctt?"
r.~ :
II
Corb jn-t^eleta"
reorrongeaent
« I
-C. -C..-
'* -C.-C.-
Glutowote
*utat«
*«thyl«alonyi-co«ncy*e
H R
R
H
A *vtett
iutarat*
•utote
N i g r otio" o* on
0* 1"o grouo
NM,M
H
1.-?!—
»uljii
D-e-lvti*e
»utote
D-or«ithin#
sctat*
*4
MQ-r, .r,,i 1 ud mO-C
C o " v o n l o " of
d i o l t o r o«ino
L-f-ly»»«e
Dioldehydrote
*1r?.
Glycerol
X
x
dehydrate
M
o l c o h o l t to
i 1
oidohydot
*
O e C - £ „ - ♦ *H
o«*enia-lyot«
|2" •
M
■* ri00" o cl>-:Ud«t to 2 ' - dec. r
8.
c 1»■-». i i t *
Re-?c tic*
Bote ^ O ^ H ^ Q P P P
Bote y O . C H . O P P P
Ri bo nueleotid*
roductote
(RSH)j
Q
O H OH
OH
♦ («S)2 ♦ >y>
(* S M ) 2 . dithiol
Clott 2 - Reoctiont
involving mothylcobalgwjn
Reaction
5 - C H j -THF ♦ H S ( C H 2 )2 C H ( N H 2 ) C O O H
^
N^-methy 11 e t r o hydrofoloto:
THF ♦ C H . S ( C H ' C H ( N H j C O O H
J
2 Z
2
ho«oeyitoino mothyltron*f e ro te
3 - C H 3 - THF ♦ H S ( C H 2 )2 S 0 3 — ) THF ♦ C H 3 $ ( C H 2
"Methone tynth.tate"
5- C H 3.THF ♦ COj ■
THF * t e t r a h ) d r o f o l a t e
y THF ♦ C H 3 C O O
"Acetate tynthotato"
10
reported (e.g. Pratt, 1984; Finke and Hay, 1984).
In this work only the
isomerase
reactions are considered.
In
Chapter 4, model studies with protein-free cobalt corrinoids aim to
clarify the rflle of the protein in isomerase enzymes.
For a recent
review of model studies concerning isomerase enzymes see Finke
et
al. (1984).
TABLE 1.1
Enzyme
reactions
requiring cobalt corrinoids
Chemaly (1980)).
Clot* 1 - S«actior» requiring o d c n o t y i c o b o l o m i n
Ca r b o n . t « « l # t o «
rtarrong«*«nt
H
R
*
H
Gluto*ot« «utoi«
thy I m o l o n y i - c o o n Xy«*
A Mgtot*
«-H*tHylon«glwtarot*
■uttiM
C onvortion of
d iolt or a«ino
o l c o h o l t to
oidohydct
M
-» r i b o n u c ’, 0 ; i j,, »c 2 ' - duo , r ibe-v c > ■
;♦
.i U %
8 .
0 * o c t ion
*ibonucl«otid*
raductoi*
O H OH
OH
♦(RS)2 ♦H20
( R S H )2 . dithiol
Clou
2 - W#oction« involving
faction
THF»t«trohydrofolate
leobolowin
(source:
11
1.3
Iron porphyrins used in this work
1.3.1
Cytochrome c
(a ) History and description
The cytochromes are a group of haemoproteins whose principal
biological
function
molecular
oxygen
is electron and/or hydrogen
in
reversible valency
the
respiratory
change
Union of Biochemistry,
of
chain,
their
haem
transport
by
iron
virtue
of
to
a
(International
1961). Cytochromes also play a role
in
photosynthesis (Hill, 1954) and in non-photosynthetic anaerobic
and chemosynthetic bacterial reactions. Keilin first Identified
three different types of cytochrome (cytochromes a, b, and c)
(Keilin,
1925;
1926) and showed that the respiratory
of cytochromes was related
Iron.
Ir
fact,
distinguished
four
on
to a
main
the
valency
groups
if
basis
change
of
on
function
the
cytochromes
their
prosthetic
hsem
can
be
groups:
cytohaems a, b, c and d are shown in Figure 1.6. Cytochromes c
are
unique
in
covalently
that
bound
to
the
prosthetic
the
peptide
cysteine
residues
on
typically
transfer
electrons
themselves
or
the
other
group,
by
cytohat-m
thioether
polypeptide
chain.
in multienzyme
molecules
su'-h
c,
is
linkages
to
Cytochromes
systems
as
the
between
cytochrome
reductases, dehydrogenases or the cytochrome peroxidases. This
is illustrated in Figure 1.7 for the mitochondrial
chain:
the rflle of cytochrome c
cytochrome
c
reductase
and
in electron
cytochrome
c
respiratory
transfer between
oxidase
is
clearly
indicated.
Cytochrome
(1925);
its
(Mf ^
was
wide
invertebrates
small
c
first,
occurrence
and y* ast
12 000)
named
in
has since
monohaem
and
described
cells
been
protein,
oxidation chain in the mitochondria,
from
by
Keilin
mammals
established.
It
part
terminal
of
the
between
the
ferric
and
is
a
which transfers electrons
from cytochr >me c reductase to cytochrome c oxidase. The
alternates
to
ferrous
states
iron
with
a
midpoint potential at pH 7 of 260 ♦ 20 mV (Lemberg and Barrett,
1973). The complete amino acid sequence is known for at least
thirty-six species
(Lemberg anu Barrett, 1973, p. 13G) ranging
12
C(»M||0
CMj
I
'0 M
CM,
CH.
i
CM.
I
M,
CM,
....................
OCM
p
^ T 'O '
CO.M
CO.M
“
CM,
CM,
CM.
I
CM,
I
M.
,CM,
*-,cSc#e.c ,c;c,e
M
M,
CM.
M
M*«m «(CvlohMm «>
n
c *»
m c 'om
M
CM,
Cm .
CM,
cm
/
M.C
.
M.C
M .c
M>«
CO.M
CO,M
CO.M
Pr»«*fc.«NW(C
4).
C O lH
C<^H«n 4
— CO
s NM
I
M
s, 8
,C0 -NMV
M *C ' C m
Fig. 1.6
Prosthetic groups of
the
cytochromes
(source:
Lemberg
und Barrett (1973)).
(I)
subtlraiui
succimio
II
*Ilit h *|mi »it«tr> <liitm
( ’ ) |I|( I m m i I H % l l l l t l t I l l l t t *1 . l l l l l
I . II,
III,
I V
I lie
In in
M l » ) » H I I* M i l I M l l l t l | « >1 l * V
in n i| tli)iv i
lli.il
tA ii
M ill'll H |»llll
l it * i h u I i i U i I ( I ) , ( 2 ) . (
lllll'
lin t l l i i i t
m
I i * • »f p l t n t *
p l l i n ) l.llH M I.
Fig. 1.7
Cytochrome
c
in a multienzyme
and Barrett (1973)).
system
(source:
Lemborg
from mammala and birds to reptiles,
studies
have
considerable
amphibia and fishes. These
evolutionary
significance.
The
tertiary folding of cytochrome c has been maintained at
since
mammals
and
fishes
diverged,
400
million
least
years
ago
yeast
was
(Dickerson et a l ., 1971).
(b ) Structure
The preparation of cytochrome c from ox-heart
pioneered by Keilin, Hartree and Theorell
and
in the 1930's
(e.g.
Keilin, 1930). The tertiary structure of horse-heart cytochrome
c was established by Dickerson and Margoliash (e.g. Margoliash
0
et al., 1960), with 4 A resolution. A very close analogue of
this
cytochrome,
with
tryptophan
59
substituted
phenylalanine has been synthesized (Sano and Kurihara,
by
1969).
a
An X-ray crystallographic structure with 2.8 A resolution was
reported by Dickerson (1971)
for the same cytochrome specie*..
Recent X-ray crystallographic studies
(Takano
1961a;
X-ray
1961b)
structure)
conducted
and
EXAFS
measurements
with
both
(extended
(Labhardt
ferric
and
and
and
Dickerson,
absorption
have
Yuen,
ferrous
fine
cytochrome
been
c.
The
structures deduced from X-ray diffraction were confirmed by NMR
(nuclear magnetic resonance) studies to be largely conserved in
solution
(Moore
and
Williams,
1980).
The
structure
of
cytochrome c is shown in Figure 1.8. The X-ray studies confirmed
the inclusion of the haem prosthetic group in a crevice of the
protein
molecule,
as
postulated
(1955a). A polypeptide chain of
by
Ehrenberg
Theorell
104 amino acids wraps around
the hatim peptide in two halves, residues 1 and residues 48 - 91 to th«a left of
spin haem c, which
and
to the right
the redox centre,
a low
is recognised as the organising principle
(Lemberg and Barrett,
1973; Dickerson et al.,
1971). The haem
O
is contained sideways
length), with one
bridge 0),
over
edge,
exposed
the top of
to
in the resultant crevice
the solvent.
and
the
in
residues.
Residues 94 - 104 return
to the right and are
sulphur
of
the
only
Four of the ligands to the haem
iron are from the porphyrin itself. Histidine
ligand,
21 A
between pyrrole rings II and I K m e t h i n e
the molecule
genuinely a-helical
(ca.
methionine
18 is the fifth
80, the sixth.
The
Fig. 1.8
The structure of cytochrome c (source:
Dickerson et a l .
binding of methionine 80, postulated by Harbury et a l . (1965)
and supported by a model study (Shechter and Saludjian,
and NMR
studies
(e.g.
WUtr ich
(1969)),
was confirmed
1967)
by
the
bind
via
X-ray study.
Histidine
18
and
cysteines
thioether linkages to
2)
and
II
(position
the
4),
14
porphyrin
and
17,
which
(pyrrole rings
respectively)
extend
I (position
from
the
right
wall of the crevice. Methionine 80, which extends from the left
wall,
forms part of a
(67 - 82)
which
largely
includes
invariant sequence of
lysine
residues
72,
channels filled wit.h hydrophobic side-chairm
73 and
residues
79.
Two
le.id to the right
and left from the haem to the surface, and each is surrounded
by a cluster of positively charged c-amino groups
residuee,
where
it meets
the
surface.
molecule between the positive regions,
negatively charged acidic groups.
been
implicated
in
the
At
the
rear
lysine
i>/
the
lies a cluster of nine
These
surface
of
cytochrome
linkages
of
features have
c
to
other
enzymes.
( c ) Electron transfer and conformational change
Cytochrome
c
interacts
cytochrome
oxidase
with
both
complexes
in
cytochrome
the
reductase
terminal
and
mitochondrial
oxidation chain, in complex reactions (Moora et a l ., 1982). The
reductase and oxidase molecules are additionally coupled to ATP
synthesis
and
the oxidase
also redices oxygen to
water.
The
Interaction site on cytochrome c for the oxidase and reductase
complexes appears to be largely the same for both complexes and
includes
the exposed edge of
the haem
(Rieder
tind flosshard,
1980). The electron transfer site is probably the exposed edge
of the haem
(Moore et al.,
1962)
where
protein
residues are
relatively mobile. Poulos and Kraut (1980) have suggested that
ph-nylalanine 82 forms part of the electron
The electron
transfer
function has
been
transfer pathway.
modelled
with
iron,
cobalt and ruthenium hexacyanides (Ragg and Moore,
1984) where
the
and
interaction
electron
site
transfer
is
the
probably
exposed
occurs.
haem
An
edge
oxidation
direct
linked
conformational change of the cytochrome c protein on the basis
of differences in the ferric and ferrous species was suggested
as early a* 1913 (Zelle and feuter, 1933). Reduced cytochrome c
appears to be more compact and rigid than the oxidised species.
It is less susceptible to digestion by trypsin (Okunuki et al.,
1965)
and shows a greater thermal stability than the oxidised
molecule
(Butt
and Keilin,
1962). The methionine bond to haen
iron, which in ferric cytochrome c is relatively easily broken
0
by pH or increasing temperature (Angstrom et a l ., 1982), is
particularly stable in the ferrous species., even at extremes of
temperature and pH (Moore et a l ., 1982). No denatured protein
where
the methionine-iron
lyophilised
ferrous
bond
cytochrome
has
c,
been
as
it
brokan
is
for
is
fo^nd
the
in
farric
spocies
(Avirtn and Schejter,
1972).
Hefined X-rav structures
of bonito cytochrome c have confirmed
between
the
two
redox
states
that dtfferenceB exist
(Takano
ind Dickerson,
198:a;
1981b).
Thus, cytochrorm
the
oxidised
c is far more ste-.t e in the
state;
the
environment also serves
the
ferric
r»tate.
hydrophobic
to stabilise
In
consequence,
relatively high (260 ♦ 20 mV)
lured than
nature
th*
tr - ferrous
the
re >x
(Lemberg and
nature of the conformational change is
reli<-ive to
: otential
ot k own for
protein are likely (Moore et a l ., 1 9 8 2 1
X-raj
studies
has
been
tain,
movement
o'.' the
haem
group
the
1981a;
and
in
structure
is
at
the
axial
mcludn
g&nds,
th-
of
th-
isoleucine 57. This is supported ty
*n«*
some
-he
gr ups
tyrosine 67).
1980) agr*
back
conformatio-.al
“ -:ri : it
moving by up to 0.07 nm (asparagine 52 ar
and NMP data (Moore et al.,
jf the
ietailed model based
proposed
change ^Takf;no and Dickerson,
is
19"’
’
J . The
Barrett,
but minor changes in structure extending -o tne surface
on
haem
-ray
major
•»
Ue,
-
U
nge
clo**-
modif:
to
-ion
studies of Bosahard and ZLirrer (19*<’ .
The
role
of
the
methionine-iron
linked conformational change remains
(Moore
et
al.,
cytochrome
c
1980)
of
reported
b
the
o n — --- - .a l . NMP studies
cobalt, yr- n r — -
a
smal
oxidation-
incrwta*
c
ir
and
metal-sulphur
interaction, on reduction, interpvafead aa a b m d length
But
X-ray
crystallographic
atucj es
1981a; 1981b) and EXAFS m e a s u r e ^
and Yuen,
1979)
and model
contradict
the
properties"
(Moore
et
-s on
complexes
assertion.
al.,
(Takar
-yt
19*.')
and
1
■
Dickerson,
nr- me c iLjbhardt
(Mash *
H wever,
native
et
al.,
inomaloua
d i s p l a y - 1 by
the
198; ',
dynamic
bond
in
ferricytochrome merit its further investis»-ion.
(d ) The haem ir«m-methion in»» bond
The methionine ligand of cytochrome c is displaced by cyanide,
azide and imidazole at pH 7 (Stellwagen,
Redfield,
complexes,
1970;
Sutin
and
Yandell,
and by a residue at ca.
1966;
1972)
1968; Gupta and
producing
pH 9.0,
the
low-spin
identity
of
which has not definitely been established (Bosshard, 1981). The
Author Aron Janine
Name of thesis The Coordination Chemistry Of Some Cobalt Corrinoid And Iron Porphyrin Complexes. 1986
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