principles of bioinorganic chemistry - 2004
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Principles of Bioinorganic Chemistry - 2004
The grade for this course will be determined by a term exam (35%), a written research paper with oral presentation (55%), and problem sets (10%). The oral presentations will be held in research conference style at an all-day symposium at MIT on Saturday, October 30th. Please reserve the date for there are no excused absences. Papers are due October 28th. Problem sets are due one week after their assigned date. Recitations are held at 5 PM on Mondays.
WEB SITE: web.mit.edu/5.062/www/
Lecture Date Lecture Topic Reading Problems1 9/9 (Th) Intro; Choice, Uptake, Assembly of Mn+ Ions Ch. 5 Ch. 12 9/14 (Tu) Metalloregulation of Gene ExpressionCh. 6 Ch. 23 9/16 (Th) Metallochaperones; Metal Folding, X-linking.Ch. 7 Ch. 34 9/21 (Tu) Med. Inorg. Chem./MetalloneurochemistryCh. 8 Ch. 45 9/23 (Th) Mössbauer, EPR, IR Spectral FundamentalsCh. 9 Ch. 56 9/28 (Tu) Electron Transfer; Fundamentals Ch. 9 Ch. 67 9/30 (Th) Long-Distance Electron Transfer Ch. 10 Ch. 78 10/5 (Tu) Hydrolytic Enzymes, Zinc, Ni, Co Ch. 109 10/7 (Th) CO and Bioorganometallic Chemistry TBA Ch. 810 10/12 (Tu) Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 11 Ch. 911 10/14 (Th) O2 Activation, Hydroxylation: MMO, ToMOCh. 11 Ch. 1012 10/19 (Tu) Model Chemistry for O2 Carriers/ActivatorsCh. 12 Ch. 1113 10/21 (Th) Complex Systems: cyt. oxidase; nitrogenase Ch. 12 Ch. 1214 TBA Term Examination
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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
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Properties of Transferrin
Glycoprotein, Mr = 80 kDa; Kapp = 1020 M-1 Fe3+ and CO32- bind synergistically.
Protein has two domains. In each domain there are two subdomains that clamp
down on the iron and carbonate ions.
Note hinge motion that accompanies iron/carbonate binding
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Transferrin and Structural Changes on Fe Binding
Baker, Anderson, and Baker, PNAS, 2003, 100, 3579.
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Transferrin Active Site Geometry
Tyr
HisAsp
Tyr
Arg
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.
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O
C
O
H2C
C
H
+H3N COO-
O
C
O
NHH2C
C
H
+H3N COO-
CH2
CH2
H2C
O
C
O
CH2H2C
C
H
+H3N COO-
O
C
O
OH
Biologically available carboxylates:
-
Bicarbonate
Aspartate (Asp) D
Carboxylate Ligation in Metalloproteins
Glutamate (Glu) E
- -
Lys* Carbamate
-
Carbonate is encountered in transferrinLys* is found in urease, rubisco, and phosphotriesterase
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Various Anions Can Bind TransferrinVarious Anions Can Bind Transferrin
Crumbliss, et al. PNAS, 2003, 100, 3659.
Nomenclature: Fbp, ferric binding proteinsn, 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.
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Mechanism of Transferrin Uptake and Iron Release in Cells by Receptor-Mediated Endocytosis
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Metal Regulation of Gene Expression
PRINCIPLES:•Metal-mediated protein structure changes affect transcription•Metal-mediated protein structure changes affect translation•Apo vs holo metalloproteins bind DNA/RNA differently•Metalloregulatory protein is the sensor - inorganic chemistry•Metal-induced protein structure changes also activate enzymes•Metal-induced protein structure changes are metal-specific
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
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Regulation of Iron Levels in CellsThe 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 previouslyMetalloregulation:•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 mRNA Proteintranscription translation
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Ferritin Subunit and Channel Structure
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Ferroxidase Center Loads Fe
into ApoFt
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Mixed-valent polyiron oxo cluster prepared as a model for ferritin core formation intermediates.
Overall formula: [Fe12O2 (OCH3)18(O2CCH3)
6(CH3OH)n]
Taft, et al., Science 1993, 259, 1302
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Reminder: Apo (left) and Holo (right) Forms of TransferrinOnly Iron-Loaded Transferrin Binds to the Receptor
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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.
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IRP
IRP
Components of the Metalloregulatory System
Stem-loop
structure in the
mRNA
Iron-responsive
protein (IRP)
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IRP
IRP
Regulation eventsHigh Fe, low TfR, high FtLow Fe, high TfR, low Ft
Message translated Message degraded
Message blocked Message translated
Ferritin Transferrin
Fe
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IRP1 is the Cytosolic AconitaseContains an Fe4S4 Cluster
Cluster assembled inprotein, which then dissociates
frommRNA
S
SFe
SFe
Fe
SR
RS
RS
SR
Fe
S
Apoprotein stays associated with
mRNA
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Regulation of a Toxic Metal, MercuryThe 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 lyaseMercuric ion reductaseMerR, the intracellular mercuric ion sensor
The implications:Transcription of the genes encoding the
proteins is controlled by MerR in response to mercury
levels
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merT merA merB
The Mercury Resistance Operon: Genes and Protein Functions
merB encodes an organomercurial lyase (under control of merR operon):
RHgX + H+ + X- organomercurial lyase RH + HgX2
merA encodes a mercuric ion reductase (under control of merR operon):
HgX2 + NADPH + H+ Hg(0) + NADP+ + 2RSHmercuric ion
reductaseX- = RS-
Turnover rate, 1 - 100 mol min-1
Slow, but still 106 x spontaneous reaction
Mr, 22 KDa
Hg(0) is non-toxic and volatile
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Postulated Mechanism for Organomercurial Lyase
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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 consistentwith the thermodynamic stability of [Hg(SR) 3]- complexes and may account both for the high affinity of the Hg(II) binding and forthe selectivity for Hg(II) over other soft metal ions thatprefer tetrahedral metal-thiolate coordination.
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Effect of [Hg2+] on Transcription Activity