Study exercises for the 2nd Partial Exam

Yachay-Tech School of Chemical Sciences and Engineering

Bioinorganic Chemistry
Author: Juan Pablo Saucedo, PhD
Alfredo López - Hugo Rey- Kenia Luzuriaga - Samuel Vallejo- Jordy Cusangua
Fill the table with the information about the spectroscopies

Spectroscopic Electromagnetic Selection Rules Example

Technique radiation
EPR 10-4 to 10-1 m Δms = ±1 EPR spectra of iron 3+
Δml = 0 species. Particularly, Fe (III) of
siderophores; ferrichrome.
UV-Vis 100 to 800 nm Spin: Δs= 0 Uv-vis spectra of chlorophyll
Laporte: (photons and synthetic
u→g, g→u pigments).
Δl = ±1
CD 170 to 300 (100 to Spin: Δs= 0 Cd of Myoglobin
800) nm Laporte:
u→g, g→u
Δl = ±1
MCD 300 to 2000 (100 Right PL: Δms = +1 MCD spectra of
to 800) nm Left PL: Δms = -1 metalloporphyrins

Spin: Δs= 0
Laporte:
u→g, g→u
Δl = ±1
NMR Radio frequency Δml = 0 determine structure of
109 to 1011 nm proteins, amino acid profile,
carotenoids, organic acids,
lipid fractions, the mobility of
the water in food

1) For the following metalloenzymes, describe the composition and geometry of the
active site, as well as the reaction(s) these enzymes can perform

a) Transferrine and siderophores

Both enzymes have functions in the transport of iron which only differs on their ligands for
its whole structure. To begin with, transferrin enzymes are membrane receptors that
regulate the iron supply to cells. They can be found in plasma which has the role of
transporting iron in the bloodstream which then are further liberated into cells. Moreover,
this enzyme also provides bacteriostatic and antioxidant protection.

Its conformation ranges from di and mononuclear iron metal center which can also be
divided into 5 types of transferrins enzymes. Those are: transferrin, lactoferrin,
ovotransferrin, melanotransferrin, and ferric binding protein. For an external view, diferric
transferrin are made of 2 lobes. Each lobe has 2 domains which embed the iron metal
center. Particularly, the active site of transferrins is made of two tyrosines, one aspartate,
one histidine, and one bidentate ion carbonate as can be seen in the following figure
which in total produce an octahedral structure.

On the other hand, siderophores are enzymes that have higher affinity to Fe (III) metal
specie which produce an important complex being useful on its transportation in microbes
and its further liberation by redox reaction. Siderophores differ on its structure by the
usual ligands that they can have. Siderophores are classified according to those ligands
which are: catecholates, hydroxamates, and carboxylates.
(A) hydroxamate siderophore: desferrioxamine; (B) catecholate siderophore: enterobactin;
(C) carboxylate siderophore: vibrioferrin; (D) mixed siderophore: yersiniabactin; (E)
phytosiderophore: mugineic acid.

Usually, hexadentate ligands are what has more affinity to Fe (III) and what is usually made
by the 3 previous types of siderophores. In addition, bidentate, tridentate, and
tetradentate ligands also appear with affinity of binding over the iron species. Bidentate
ligands produce particular stereochemistry as shown in the next figure being the lambda
conformation which mostly predominates.

b) Ferredoxin, rieske and HPIP proteins

These enzymes have in common to have Fe-S clusters, which are molecular assemblies of
iron and sulfide, in their active sites. Besides, these types of enzymes are employed as
electron carriers, they are also known to perform functions as reduction of disulfide bonds
and initiation or stabilization of radical chain reaction or serving as Lewis’s acids1. These
types of enzymes are classified depending on the type of Fe-S cluster in their active site,
thus we have: ferredoxins (low-potential [2Fe–2S], [4Fe–4S] and [3Fe–4S]), Rieske
proteins (which are high-potential [2Fe–2S] proteins), and high-potential iron–sulfur
proteins (HiPIPs, which are high-potential [4Fe–4S] proteins).2

Ferredoxin

As we mentioned before Ferredoxins can be subdivided in different categories depending

on their type of Fe-S cluster as well; the first family to review are those with low potential
[2Fe-2S] cluster, which as their name suggest these enzymes contains as their active site
two iron atoms that are coordinated by two inorganic sulfurs and four cysteine thiolates
from the protein in a distorted tetrahedral arrangement as we observe in the following
image. Besides, these Ferredoxins are one-electron carriers’ enzymes whose function is
largely unknown, although some data suggest an involvement in nitrogen metabolism.

Secondly, in Ferredoxins we can found enzymes with [4Fe-4S] clusters, which contains four
iron atoms where each one is coordinated with three inorganic sulfurs and one cysteine
thiolate taking the full arrangement the form of a distorted cube with iron and sulfur
atoms positioned alternatively in the apexes. These enzymes [4Fe−4S] clusters are known
to be the first clusters formed in the early earth environment and are important in
hydrogen evolution in anaerobic bacteria, in which the reduced form of ferredoxin
transfers electrons to H+ as the final acceptor or they can be in the more reducing parts of
photosynthetic and aerobic electron transfer chains.

Finally, in Ferredoxins there are also enzymes with clusters of the type [3Fe-4S], which can
be seen as a cube of the [4Fe-4S] cluster with one iron missing by any other reaction as
oxidative damage. Thus, [3Fe−4S] clusters are asymmetric in comparison with the before
one. These enzymes are found exclusively in bacteria, mainly anaerobic bacteria, and are
involved in anaerobic metabolism and also, they have been reported to can act in sulfite
reduction.

Rieske protein

These types of proteins have the [2Fe-2S] type of Fe-S cluster, but these are distinguished
from the Ferredoxin analogs by their unique His2-Cys2 ligation motif. Thus, the active site
consists of two irons coordinated each one with two inorganic sulfurs and the iron atom
closer (2+) to the surface has histidine ligands and the other iron (3+) is bound to two
buried cysteine ligands. These enzymes were discovered by Rieske in 1964 and are found
in bc complexes of mitochondria and bacteria, or complexes of chloroplast. Rieske protein
is responsible for hydroquinone oxidation and acts as the first electron acceptor.
HPIP protein

The high-potential iron proteins (HiPIP) are small proteins isolated from photosynthetic
bacteria. The active site of these enzymes consists of a high potential [4Fe-4S] cluster,
however, the higher reduction potential of HiPIPs results in one less electron in both the
reduced and oxidized states of these proteins compared to ferredoxins, meaning a
[4Fe−4S]2+,3+ state. The function of HiPIP enzymes is as electron donors to the tetraheme
cytochrome in photosynthetic bacteria.

c) P450 cytochrome

Necessary to dioxygen activation by iron proteins mechanism.

1)Ferric heme state is a 6-coordinate, low-spin species, substrate binding to the active site
pocket displaces the aqua ligand and turns the heme into a high-spin 5-coordinate iron
complex with an increased redox potential.

2) Reduction of the high-spin ferric heme, usually by nicotinamide adenine dinucleotide

generates a high-spin F 2+ heme, which is activated for O2 binding.

3) Addition of O2 gives a ferric superoxo complex.

4) A second reduction event occurs to give a ferric peroxo complex, which is then
protonated to form a ferric hydroperoxo intermediate labeled Compound 0.

5) Heterolytic cleavage of the O–O bond of Compound 0 generates a high-valent iron–oxo

porphyrin species, F e-iv, labeled Compound I.
6) Compound I abstract an H-atom from the C–H substrate to form Fe 4+ or protonated
Compound II.

7) Carbon radical rapidly recombines with protonated Compound II to give an alcohol

product and the ferric heme resting state.

d) Cupredoxins, plastocyanine, Superoxide Dismutase (SOD)

Cupredoxins are small proteins containing type I copper centers,

which are ubiquitous in nature. They function as electron transfer
shuttles between proteins. These proteins may have evolved to
bind specifically to copper, develop recognition sites for specific
redox partners, tune the redox potential for a particular function,
and allow for efficient electron transfer through the protein matrix.
Ligands: Metionina, 2 Histidina y Cisteína. Tetrahedral geometry.

Plastocyanin (PC) is a β-sheet protein with a Cu atom that acts as the redox center. The Cu
atom is coordinated to four amino acid residues (His87, His37, Cys84, and Met92) in a
distorted tetrahedral geometry. The distorted tetrahedral geometry of the Cu center
together with the protein matrix promotes efficient electron transfer in the photosynthetic
reaction of plants.
Superoxide Dismutase (SOD) (Type-2 copper) of copper and zinc contains a Cu(II)-Zn(II)
heterodinuclear center with imidazolate bridge in its active site. The copper ion is bound
to three histidines, one water molecule and to the imidazolate bridge, whereas the zinc
ion is bound to two histidines, one aspartate, and to the imidazolate bridge. The copper
ion is in a distorted square-pyramidal geometry, while the zinc ion located at a distance of
6.2 A from the copper ion is in a distorted tetrahedral structure. Its function is to catalyze
the disproportionation of the superoxide ion.

e) Alcohol dehydrogenase

They are a group of dehydrogenase enzymes that occur in many organisms and
facilitate the interconversion between alcohols and aldehydes or ketones with the
reduction of nicotinamide adenine dinucleotide (NAD+) to NADH. Additionally, it
provides a line of defense against a common toxin in our environment. Instead, when
talking about geometry, the structural zinc site
is composed of four closely spaced cysteine
ligands arranged in a nearly symmetric
tetrahedron around the Zn ion. A recent study
showed that the interaction between zinc and
cysteine is mainly governed by an electrostatic
contribution with an additional covalent
contribution to the bond.
Likewise, within the subunits involved, the
substrate is coordinated with zinc and this
enzyme has two zinc atoms per subunit. One is
the active site, which is involved in catalysis. At the active site, the ligands are Cys-46,
Cys-174, His-67, and a water molecule.

There are five classes (I-V) of alcohol dehydrogenase, but the hepatic forms that are
used primarily in humans are class 1. Class 1 consists of α, β, and γ subunits that are
encoded by the genes ADH1A, ADH1B, and ADH1C.[22][23] The enzyme is present at
 high levels in the liver and the lining of the stomach.[24] It catalyzes the oxidation of
ethanol to acetaldehyde (ethanal)

f) Zinc fingers
Zn2+ is intermediate in ‘‘hard/soft’’ acid characteristics and the most common amino
acid binding sites for tetrahedral zinc come overwhelmingly from a combination of
histidine (N-donor), glutamate or aspartate (O-donor) and cysteine (S-donor) residues,
and all three ligands can bridge one or several zinc ions. In broad terms, the
intermediate to fast ligand exchange, stereochemical flexibility, and redox-inertness of
zinc are additional key features enhancing the use of the ion for the function of so many
proteins and diverse processes.Structural zinc covers an equally important area and is
described by ligation of four protein derived histidine and cysteine ligands yielding a
coordinatively saturated central metal ion and prominent amongst these sites are the
zinc fingers. The diverse range of biological functions of zinc-fingers and zinc-finger-like
proteins includes DNA recognition, RNA packaging, transcriptional activation, regulation
of apoptosis, protein folding and assembly, contributing to the manifestation of
fundamental cellular processes such as development, differentiation, and tumor
suppression.The zinc fingers are generally classified according to the number and type
of amino acids involved in the Zn(II) coordination, such as Cys2His2, Cys3His or
Cys2HisCys