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CHEMISTRY OF
METALLOPROTEINS
WILEY SERIES IN PROTEIN AND PEPTIDE SCIENCE
Flexible Viruses: Structural Disorder in Viral Proteins • Vladimir Uversky and Sonia
Longhi
JOSEPH J. STEPHANOS
ANTHONY W. ADDISON
Copyright 2014 by John Wiley & Sons, Inc. All rights reserved
Published by John Wiley & Sons, Inc., Hoboken, New Jersey
Published simultaneously in Canada
No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form
or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as
permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior
written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to
the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400,
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should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken,
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Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts
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Library of Congress Cataloging-in-Publication Data:
Stephanos, Joseph J., author.
Chemistry of metalloproteins: problems and solutions in bioinorganic chemistry/by Joseph J.
Stephanos, Anthony W. Addison.
p.; cm. – (Wiley series in protein and peptide science)
Includes bibliographical references and index.
ISBN 978-1-118-47044-2 (paperback)
I. Addison, A. W., author. II. Title. III. Series: Wiley series in protein and peptide science.
[DNLM: 1. Metalloproteins–chemistry–Examination Questions. QU 18.2]
QP551
5720.6076–dc23
2013041995
Printed in the United States of America
10 9 8 7 6 5 4 3 2 1
CONTENTS
Preface ix
1 Introduction 1
Proteins: Formation, Structures, and Metalloproteins, 4
References, 28
3 Nonredox Metalloenzymes 71
Carboxypeptidases, 75
Carbonic Anhydrase, 84
Alcohol Dehydrogenase, 88
References, 91
4 Copper Proteins 95
Introduction, 95
Electronic Spectra of Copper Ions, 96
ESR Spectra of Copper Ions, 105
Copper Proteins, 117
Plastocyanin, 119
Azurin and Stellacyanin, 127
Superoxide Dismutase, 131
v
vi CONTENTS
Hemocyanin, 135
Ascorbic Oxidase, 139
References, 142
7 Chlorophyll 407
References, 421
Index 423
PREFACE
This book is an attempt to reveal the chemical concepts that rule the biological action
of metalloproteins. The emphasis is on building up an understanding of basic ideas
and familiarization with basic techniques. Enough background information is pro-
vided to introduce the field from both chemical and biological areas. It is hoped that
the book may be of interest to workers in biological sciences, and so, primarily for this
purpose, a brief survey of relevant properties of transition metals is presented.
The book is intended for undergraduates and postgraduates taking courses in
coordination chemistry and students in biology and medicine. It should also be a value
to research workers who would like an introduction to this area of inorganic
chemistry. It is very suitable for self-study; the range covered is so extensive that
the book can serve as a student’s companion throughout his or her university career.
At the same time, teachers can turn to it for ideas and inspirations.
The book is divided into seven chapters and covers a full range of topics in
bioinorganic chemistry. It is well-illustrated and each chapter contains suggestions for
further reading, providing access to important review articles and papers of relevance.
A reference list is also included, so that the interested reader can readily consult the
literature cited in the text.
It is hoped that the present book will provide the basis for a more advanced study in
this field.
JOSEPH J. STEPHANOS
ANTHONY W. ADDISON
ix
1
INTRODUCTION
• A physiological deficiency appears when the element is removed from the diet.
• The deficiency is relieved by the addition of that element to the diet.
• A specific biological function is associated with the element.
1
2 INTRODUCTION
FIGURE 1-1 Distribution of elements essential for life (Cotton and Wilkinson, 1980).
INTRODUCTION 3
This section is designed to introduce the chemistry of proteins. The text broadly
includes where and how the proteins are formed, along with the structure and
formation of metalloproteins.
Following the introduction of organelles and their functions within the cell, the
discussion will be concerned with the general structure of deoxyribonucleic acid
(DNA) and how the nucleus maintains its control of cell growth, division, and
formation of [messenger, transfer, and ribosomal ribonucleic acid (mRNA, tRNA,
PROTEINS: FORMATION, STRUCTURES, AND METALLOPROTEINS 5
rRNA)]. This is followed by how mRNA and tRNA master the formation of
proteins within a cell. Then, primary, secondary, tertiary, and quaternary structures
of the formed proteins and the factors that control each of these structures are
discussed.
Specific points about the ligation of various metal ions to different amino acids
within the proteins are made, and the binding stabilities of various metal ions toward
different amino acids are arranged.
The general formulas, side chains, and corresponding names of the common
natural α-amino acids, the formation of the peptide chain from the amino acids, and
the physiological roles of proteins are described.
The chemistry of the prosthetic and cofactors is explored. Enough basic bio-
chemistry is presented to enable the student to understand the discussions that follow.
Structure of DNA
• The order in which they appear on the chain makes up the molecular message
(Fig. 1-4).
• The DNA molecule is also capable of duplicating itself and dividing.
8 INTRODUCTION
How does the nucleus maintain its control of cell growth and division?
• During ordinary cell division called mitosis, two new cells result from a single
parent.
• Each daughter has the same number of chromosomes as the parent.
• If DNA is the molecular stuff of the chromosome, it must be able to reproduce itself.
• The DNA double helix rewinds and separates into two single strands (Fig. 1-6).
• As the unwinding occurs, the single strands act as templates for synthesis of new
complementary strands.
• When the parent DNA double helix has completed its unwinding, two new
DNA double-stranded molecules are formed.
• The process by which new DNA is formed is called replication.
Protein Synthesis
• The order of the N bases on the DNA molecule determines the order of amino
acids in the protein molecule.
• While DNA is in the nucleus, the proteins are synthesized on ribosomes outside
the nucleus as follows:
FIGURE 1-6 DNA double helix rewinds and separates into two single strands.
PROTEINS: FORMATION, STRUCTURES, AND METALLOPROTEINS 11
As the DNA double helix unwinds, the N base segment becomes exposed.
The DNA molecule serves as template for the synthesis of mRNA molecule.
The synthesis of mRNA is analogous to the replication synthesis of DNA
(Fig. 1-7).
mRNA has structure similar to DNA but contains:
• Ribose instead of deoxyribose
• N-base uracil instead of thymine:
• After the tRNA has discharged its amino acid passenger, it moves out into the
cytoplasm, finds another amino acid, and returns to the ribosome surface.
Give the general formula, side chain, and corresponding name of the common
natural α -amino acids.
(continued )
14 INTRODUCTION
How can the peptide chain be formed from the amino acids?
Define: primary, secondary, tertiary, and quaternary structures. And what are
the factors that control each of these structures?
The groups of four gray-shaded atoms are coplanar. Free rotation occurs
about the bond connecting the carbon with the carbonyl and the nitrogen.
Therefore, the extended polypeptide chain is a semirigid structure with two-
thirds of the atoms of the backbone held in a fixed plane.
Examples of secondary structures:
(a) random coil
(b) α–helix (Fig. 1-9)
(c) β–pleated (Fig. 1-10), associated as (i) parallel and (ii) antiparallel
(d) reverse turns (Fig. 1-11)
(e) omega loops (Fig. 1-12)
Both reverse turns and omega loops appear at the outer surface of the
molecules.
• A Tertiary structure refers to the folding of the already secondary structured
amino acids to form a three-dimensional (3D) structure. The overall 3D
architecture of the polypeptide backbone:
Fibrous proteins: coils (Fig. 1-13).
Globular proteins: compact, ellipsoidal, spherical, until denatured. The
folded tertiary, globular, structure of myoglobins is imposed over the
helical secondary structure. Structures from X-ray diffraction are shown
in Fig. 1-14.
Synthetic polypeptides have random or simply repetitive structures.
PROTEINS: FORMATION, STRUCTURES, AND METALLOPROTEINS 17
Nevertheless, there was the hope of the seven days when his money
might be gone. One by one the seven days came, and in each one it
seemed to her in the midst of her waiting as though the day was
come for his return. She had never been a woman to gad about the
little hamlet or chatter overmuch with the other women there. But
now one after another of these twenty or so came by to see and ask,
and they asked where her man was, and they cried, “We are all one
house in this hamlet and all somehow related to him and kin,” and at
last in her pride the mother made a tale of her own and she
answered boldly, from a sudden thought in her head, “He has a
friend in a far city, and the friend said there was a place there he
could work and the wage is good so that we need not wear
ourselves upon the land. If the work is not suited to him he will come
home soon, but if it be such work as he thinks fit to him, he will not
come home until his master gives him holiday.”
This she said as calmly as she ever spoke a truth, and the old
woman was astounded and she cried, “And why did you not tell me
so good a lucky thing, seeing I am his mother?”
And the mother made a further tale and she answered, “He told me
not to speak, old mother, because he said your tongue was as loose
in your mouth as any pebble and all the street would know more than
he did, and if he did not like it he would not have them know it.”
“Did he so, then!” cackled the old mother, leaning forward on her
staff to peer at her daughter’s face, her old empty jaws hanging, and
she said half hurt, “It is true I ever was a good talker, daughter, but
not so loose as any pebble!”
Again and again the mother told the tale and once told she added to
it now and then to make it seem more perfect in its truth.
Now there was one woman who came often past her house, a widow
woman who lived in an elder brother’s house, and she had not
overmuch to do, being widowed and childless, and she sat all day
making little silken flowers upon a shoe she made for herself, and
she could ponder long on any little curious thing she heard. So she
pondered on this strange thing of a man gone, and one day she
thought of something and she ran down the street as fast as she
could on her little feet and she cried shrewdly to the mother, “But
there has no letter come a long time to this hamlet and I have not
heard of any letter coming to that man of yours!”
She went secretly to the only man who knew how to read in the
hamlet, and he wrote such few letters as any needed to have written
and read such as came for any, and so added a little to his
livelihood. This man the widow asked secretly, “Did any letter come
for Li The First, who was son to Li The Third in the last generation?”
And when the man said no, the gossip cried out, “But there was a
letter, or so his wife says, and but a few days ago.”
Then the man grew jealous lest they had taken the letter to some
other village writer and he denied again and again, and he said,
“Very well I know there was no letter, nor any answering letter, nor
has anyone come to me to read or write or to buy a stamp to put on
any letter and I am the only one who has such stamps. And there
has not come so much as a letter carrier this way for twenty days or
more.”
Then the widow smelled some strange thing and she told
everywhere, whispering that the wife of Li The First lied and there
had been no letter and doubtless the husband had run away and left
his wife. Had there not been a great quarrel over the new robe, so
that the whole hamlet heard them cursing each other, and the man
had pushed her down and struck her even? Or so the children said.
But when the talk leaked through to the mother she answered stoutly
that what she said was true and that she had made the new blue
robe on purpose for the man to go to the far town, and that the
quarrel was for another thing. As for the letter, there was no letter but
the news had come by word of mouth from a traveling pedlar who
had come in from the coast.
Thus did the mother lie steadfastly and well, and the old woman
believed the tale heartily and cried out often of her son and how rich
he would be, and the mother kept her face calm and smooth and she
did not weep as women do when their men run away and shame
them. At last the tale seemed true to all, and even the gossip was
silenced somewhat and could only mutter darkly over her silken
flowers, “We will see—as time comes, we will see if there is money
sent or any letter written, or if he comes home ever and again.”
So the little stir in the hamlet died down and the minds of people
turned to other things and they forgot the mother and her tale.
Then did the mother set herself steadfastly to her life. The seven
days were long past and the man did not come and the rice ripened
through the days and hung heavy and yellow and ready for the
harvest and he did not come. The woman reaped it alone then
except for two days when the cousin came and helped her when his
own rice was cut and bound in sheaves. She was glad of his help
and yet she feared him too, for he was a man of few words, honest
and few, and his questions were simple and hard not to answer
truthfully. But he worked silently and asked her nothing and he said
nothing except the few necessary words he must until he went away,
and then he said, “If he is not come when the time is here to divide
the grain with the landlord, I will help you then, for the new agent is a
wily, clever man, and of a sort ill for a woman to do with alone.”
She thanked him quietly, glad of his help, for she knew the agent but
a little, since he was new in the last years to those parts, and a
townsman who had a false heartiness in all he did and said.
So day had passed into month, and day after day the woman had
risen before the dawn and she left the children and the old woman
sleeping, and she set their food ready for them to eat when they
woke, and taking the babe with her in one arm and in her other hand
the short curved sickle she must use in reaping she set out to the
fields. The babe was large now and he could sit alone and she set
him down upon the earth and let him play as he would, and he filled
his hands with earth and put it to his mouth and ate of it and spat it
out hating it and yet he forgot and ate of it again until he was
covered with the muddy spew. But whatever he did the mother could
not heed him. She must work for two and work she did, and if the
child cried he must cry until she was weary and could sit down to
rest and then she could put her breast to his earthy mouth and let
him drink and she was too weary to care for the stains he left upon
her.
Handful by handful she reaped the stiff yellow grain, bending to
every handful, and she heaped it into sheaves. When gleaners came
to her field to glean what she might drop, as beggars and gleaners
do at harvest time, she turned on them, her face dark with sweat and
earth, and drawn with the bitterness of labor, and she screamed
curses at them, and she cried, “Will you glean from a lone woman
who has no man to help her? I am poorer than you, you beggars,
and you cursed thieves!” And she cursed them so heartily and she
so cursed the mothers that bore them and the sons they had
themselves that at last they let her fields be, because they were
afraid of such powerful cursing.
Then sheaf by sheaf she carried the rice to the threshing-floor and
there she threshed it, yoking the buffalo to the rude stone roller they
had, and she drove the beast all through the hot still days of autumn,
and she drove herself, too. When the grain was threshed, she
gathered the empty straw and heaped it and tossed the grain up and
winnowed it in the winds that came sometimes.
Now she pressed the boy into labor too and if he lagged or longed to
play she cuffed him out of her sheer weariness and the despair of
her driven body. But she could not make the ricks. She could not
heap the sheaves into the ricks, for this the man had always done,
since it was a labor he hated less than some, and he did it always
neatly and well and plastered the tops smooth with mud. So she
asked the cousin to teach her this one year and she could do it
thenceforth with the boy if the man stayed longer than a year, and
the cousin came and showed her how and she bent her body to the
task and stretched and threw the grass to him as he sat on top of the
rick and spread it, and so the rice was harvested.
She was bone-thin now with her labor and with being too often
weary, and every ounce of flesh was gone from her, and her skin
was burnt a dark brown except the red of cheeks and lips. Only the
milk stayed in her breasts rich and full. Some women there are
whose food goes all to their own fat and none to child or food for
child, but this woman was made for children, and her motherhood
would rob her own body ruthlessly if there was any need for child.
Then came the day set for measuring out the landlord’s share of all
the harvest. Now this landlord of the hamlet and the fields about it
never came himself to fetch his share. He lived an idle rich man in
some far city or other, since the land was his from his fathers, and he
sent in his place his agent, and this year it was a new agent, for his
old agent had left him the last year, being rich enough after twenty
years to cease his labors. This new agent came now and he came to
every farmer in that hamlet, and the mother waited at her own door,
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