Principles of Bioinorganic Chemistry - 2003

Lecture Date Lecture Topic Reading Problems1 9/4 (Th) Intro; Choice, Uptake, Assembly of Mn+ Ions Ch. 5 Ch. 12 9/ 9 (Tu) Metalloregulation of Gene Expression Ch. 6 Ch. 23 9/11 (Th) Metallochaperones; Metal Folding, X-linking Ch. 7 Ch. 34 9/16 (Tu) Metals in Medicine; Cisplatin Ch. 8 Ch. 45 9/18 (Th) Electron Transfer; Fundamentals Ch. 9 Ch. 56 9/23 (Tu) Long-Distance Electron Transfer Ch. 9 Ch. 67 9/25 (Th) Hydrolytic Enzymes, Zinc, Ni, Co Ch. 10 Ch. 78 9/30 (MU) Model Complexes for Metallohydrolases Ch. 109 10/2 (MU) Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 1110 10/7 (Tu) O2 Activation, Hydroxylation: MMO, P-450, R2Ch. 11 Ch. 811 10/9 (Th) Model Chemistry for O2 Carriers/Activators Ch. 11 Ch. 912 10/16 (Th) Complex Systems: cyt. oxidase; nitrogenase Ch. 12 Ch. 1013 10/21 (Tu) Metalloneurochemistry/Medicinal Inorg. Chem. Ch. 12 Ch. 1114 10/23 (Th) Term Examination Ch. 12 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 drugsExamples: Cisplatin, auranofin as pharmaceuticals Cardiolyte (99mTc) and Gd, imaging agents

Enterobactin: a Bacterial Siderophore

Enterobactin, a Cyclic Triserine Lactone

A specific cell membrane receptor exists for ferricenterobactin. Release in the cell can occur by hydrolysisof the lactone, reduction to Fe(II), and/or lowering the pH.

O

O

O

NHR

NHRRHN

O

O

O

Fe3+ + ent6- = [Fe(ent)]3-, Kf = 1049

At pH 7, Kd = 10-25 since the

6 catechol groups have to be

deprotonated. Only the Δ isomer

.is found in nature

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.

See Raymond, Dertz, and Kim, PNAS, 100, 3584.

outer membrane

cytoplasminmembrane

intracellular esterase; hydrolyzes Ent, releases iron

Does not distinguishΔ from

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

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

Transferrin and Structural Changes on Fe Binding

Baker, Anderson, and Baker, PNAS, 2003, 100, 3579.

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.

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

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.

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 enzymes

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 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

Ferritin Subunit and Channel Structure

Mixed-valent polyiron oxo cluster prepared as a model for ferritin core formation intermediates.

Overall formula: [Fe12O2 (OCH3)18(O2CCH3)

6(CH3OH)n]

Taft, Papaefthymiou, & Lippard, Science 1993, 259, 1302

Reminder: Apo (left) and Holo (right) Forms of TransferrinOnly 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.

IRP

IRP

Components of the Metalloregulatory System

Stem-loop

structure in the

mRNA

Iron-responsive

protein (IRP)

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

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

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

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

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 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.

Effect of [Hg2+] on Transcription Activity