Principles of Bioinorganic Chemistry - 2003
Lecture
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Date
9/4 (Th)
9/ 9 (Tu)
9/11 (Th)
9/16 (Tu)
9/18 (Th)
9/23 (Tu)
9/25 (Th)
9/30 (MU)
10/2 (MU)
10/7 (Tu)
10/9 (Th)
10/16 (Th)
10/21 (Tu)
10/23 (Th)
Lecture Topic
Intro; Choice, Uptake, Assembly of M n+ Ions
Metalloregulation of Gene Expression
Metallochaperones; Metal Folding, X-linking
Metals in Medicine; Cisplatin
Electron Transfer; Fundamentals
Long-Distance Electron Transfer
Hydrolytic Enzymes, Zinc, Ni, Co
Model Complexes for Metallohydrolases
Dioxygen Carriers: Hb, Mb, Hc, Hr
O2 Activation, Hydroxylation: MMO, P-450, R2
Model Chemistry for O 2 Carriers/Activators
Complex Systems: cyt. oxidase; nitrogenase
Metalloneurochemistry/MedicinalInorg. Chem.
Term Examination
Reading
Ch. 5
Ch. 6
Ch. 7
Ch. 8
Ch. 9
Ch. 9
Ch. 10
Ch. 10
Ch. 11
Ch. 11
Ch. 11
Ch. 12
Ch. 12
Ch. 12
Problems
Ch. 1
Ch. 2
Ch. 3
Ch. 4
Ch. 5
Ch. 6
Ch. 7
Ch. 8
Ch. 9
Ch. 10
Ch. 11
Ch. 12
Principles of Bioinorganic Chemistry
Two Main Avenues of Study
•Understand the roles of naturally occurring inorganic
elements in biology. By weight, > 50% of living matter is
inorganic. Metal ions at the core of biomolecules control
many key life processes.
•Use metals as probes and drugs
Examples:
Cisplatin, auranofin as pharmaceuticals
Cardiolyte (99mTc) and Gd, imaging agents
Enterobactin: a Bacterial Siderophore
Enterobactin, a Cyclic Triserine Lactone
NHR
Fe3+ + ent6- = [Fe(ent)]3-, Kf = 1049
At pH 7, Kd = 10-25 since the
6 catechol groups have to be
O
deprotonated. Only the  isomer
is found in nature.
RHN
O
O
O
NHR
O
O
A specific cell membrane receptor exists for ferric
enterobactin. Release in the cell can occur by hydrolysis
of the lactone, reduction to Fe(II), and/or lowering the pH.
Structure of Vanadium(IV) Enterobactin
Scheme showing the ATP-driven uptake of ferric
enterobactin into E. coli cells through a specific
receptor in the cell membrane.
Does not distinguish
 from L
outer membrane
cytoplasmin
membrane
intracellular esterase; hydrolyzes Ent, releases iron
See Raymond, Dertz, and Kim, PNAS, 100, 3584.
Control and Use of Metal Ion Concentrations
PRINCIPLES:
•Homeostasis: maintain [M+ ] in proper range
•Detoxification: remove excess and/or unnatural metal ions
•Extracellular carriers
•Passive transport
•Ion channels/pumps
•Metalloregulation
•Binding and release of metal ions to receptors controlled
by pH and redox changes
•Ion concentration gradients - used to transmit energy
and information
Properties of Transferrin
Note hinge
motion that
accompanies
iron/carbonate
binding
Glycoprotein, Mr = 80 kDa; Kapp = 1020 M-1 Fe3+ and CO3 2- bind synergistically.
Protein has two domains. In each domain there are two subdomains that clamp
down on the iron and carbonate ions.
Transferrin and Structural Changes on Fe Binding
Baker, Anderson, and Baker, PNAS, 2003, 100, 3579.
Transferrin Active Site Geometry
Arg
Tyr
Asp
His
Tyr
Note that an arginine in the active
site forms key hydrogen bonds with
the coordinated carbonate ion,
helping to effect protein folding
around the metal coordination
sphere.
Carboxylate Ligation in Metalloproteins
Biologically available carboxylates:
O
-
O
O
-
O
O
C
C
H2C
OH
Bicarbonate
-
+H N
3
C
COO-
H
Aspartate (Asp) D
H2C
+
H3 N
C
O
-
O
O
C
C
CH2
NH
H2C
CH2
COO-
H
Glutamate (Glu) E
Carbonate is encountered in transferrin
Lys* is found in urease, rubisco, and phosphotriesterase
H2 C
+H N
3
CH2
C
COO-
H
Lys* Carbamate
Various Anions Can Bind Transferrin
Nomenclature: Fbp, ferric binding proteins
n, for Neisseria meningitidis
Iron must bind as Fe(III), or the ferric state. If reduced, a
bacterial reductase must be involved, thus affording control of
iron binding and uptake in the organism (see E1/2 values in the
table above.
Crumbliss, et al. PNAS, 2003, 100, 3659.
Mechanism of Transferrin Uptake and Iron Release
in Cells by Receptor-Mediated Endocytosis
Metal Regulation of Gene Expression
PRINCIPLES:
•Homeostasis: maintain [M+ ] in proper range
•Detoxification: remove excess and/or unnatural metal ions
•Metal-mediated protein structure changes affect transcription
•Metal-mediated protein structure changes affect translation
•Metal-induced protein structure changes also activate enzyme
ILLUSTRATIONS:
•Iron regulatory proteins (IRPs); control Ft and Tf translation
•Regulation of a toxic metal, mercury
•Zinc finger proteins control transcription
•Ca2+, a second messenger and sentinel at the synapse
Regulation of Iron Levels in Cells
The Players:
•Ferritin, the iron storage protein: 24-subunits, ~175 aa each;
has cubic symmetry; apoFt can house 1000 iron atoms in its
central core; a ferroxidase center loads the iron into the
protein
•Transferrin,
the uptake protein, discussed previously
Metalloregulation:
•In bacteria, occurs at the transcriptional level
•In mammals, the synthesis of apoferritin and of the
transferrin receptor are regulated at the level of translation,
not transcription
Central dogma of molecular biology:
DNA
transcription
mRNA
translation
Protein
Ferritin Subunit and Channel Structure
Mixed-valent polyiron oxo cluster prepared as a model
for ferritin core formation intermediates.
Taft, Papaefthymiou, &
Lippard, Science 1993, 259,
1302
Overall formula: [Fe12O2 (OCH3)18(O2CCH3) 6(CH3OH)n]
Reminder: Apo (left) and Holo (right) Forms of Transferrin
Only Iron-Loaded Transferrin Binds to the Receptor
Metalloregulation of Iron Uptake and Storage
Bacteria:
A single protein, Fur (for iron uptake regulator),
controls the transcription of genes involved in
siderophore biosynthesis. Fur is a dimer with subunits
of Mr 17 kDa. At high iron levels, the Fur protein has
bound metal and interacts specifically with DNA
repressing transcription.
Mammals:
Expression of ferritin and the transferrin receptor
is regulated at the translational level.
Components of the Metalloregulatory System
Stemloop
structure
in the
mRNA
IRP
IRP
Ironresponsive
protein (IRP)
Regulation events
High Fe, low TfR, high Ft
Low Fe, high TfR, low Ft
Fe
IRP
Message translated
Message degraded
Ferritin
Transferrin
IRP
Message blocked
Message translated
IRP1 is the Cytosolic Aconitase
Contains an Fe4S4 Cluster
Cluster assembled in
protein, which then dissociates from
mRNA
RS
SR
S
Fe
Fe
S
Fe
S
Fe
RS
SR
S
Apoprotein stays associated with
mRNA
Regulation of a Toxic Metal, Mercury
The problem:
Mercury in the environment of industrial plants is
converted by bacterial to harmful organomercury
compounds. Fish and other plant and animal life
assimilate the mercury which ultimately enters the
human food chain. Bacteria defend themselves
against
the mercury by using the proteins listed below.
The players:
Organomercurial lyase
Mercuric ion reductase
MerR, the intracellular mercuric ion sensor
The implications:
Transcription of the genes encoding the proteins is
controlled by MerR in response to mercury levels
The Mercury Resistance Operon: Genes and Protein Functions
merT
merA merB
merB encodes an organomercurial lyase (under control of merR operon):
Mr, 22 KDa
RHgX + H+ + X-
organomercurial lyase
RH + HgX
2
-1
Turnover rate,1 - 100 mol min
Slow, but still 106 x spontaneous reaction
merA encodes a mercuric ion reductase (under control of merR operon):
mercuric ion
HgX2 + NADPH + H+
Hg(0) + NADP+ + 2RSH
reductase
X = RS
Hg(0) is non-toxic and volatile
Postulated Mechanism for Organomercurial Lyase
MerR and Mercuric Ion Reductase Properties
Reductase: no structural or detailed mechanistic information
MerR
EXAFS spectroscopy and chemical modification experiments
indicate that Hg-MerR has a 3-coordinate, Hg(S-Cys)3
environment with an average Hg–S distance of 2.43 Å.
This unusual tridentate heavy metal receptor site is consistent
with the thermodynamic stability of [Hg(SR) 3]- complexes and
may account both for the high affinity of the Hg(II) binding and for
the selectivity for Hg(II) over other soft metal ions that
prefer tetrahedral metal-thiolate coordination.
Effect of [Hg2+] on Transcription Activity