Table Of ContentBiophysics of Electron Transfer
and Molecular Bioelectronics
ELECTRONICS AND BIOTECHNOLOGY
ADVANCED (EL.B.A.) FORUM SERIES
Volume 1 FROM NEURAL NETWORKS AND BIOMOLECULAR
ENGINEERING TO BIOELECTRONICS
Edited by Claudio Nicolini
Volume 2 MOLECULAR MANUFACTURING
Edited by Claudio Nicolini
Volume 3 BIOPHYSICS OF ELECTRON TRANSFER AND
MOLECULAR BIOELECTRONICS
Edited by Claudio Nicolini
A Continuation Order Plan is available for this series. A continuation order will bring delivery
of each new volume immediately upon publication. Volumes are billed only upon actual
shipment. For further information please contact the publisher.
Biophysics of Electron Transfer
and Molecular Bioelectronics
Edited by
Claudio Nicolini
Institute ofB iophysics
University ofGenoa
Genoa, Italy
Springer Science+Business Media, LLC
Library of Congress Cataloging-1n-Publicat1on Data
81ophysics of electron transfer and molecular b1oelectronics 1 edited
by Claudio N1col1ni.
p. cm. -- CElectronics and biotechnology advanced CEL.B.A. 1
forum series , v. 31
Includes b1bl1ographical references and index.
ISBN 978-1-4757-9518-9 ISBN 978-1-4757-9516-5 (eBook)
DOI 10.1007/978-1-4757-9516-5
1. Metalloproteins. 2. Charge exchange. 3. Molecular
electronics. I. Nicolinl. Claudio A. I!. Ser1es, Electronics and
biotechnology advanced foum ser1es , v. 3.
RC552.M46856 1998
572 · .43--dc21 98-31320
CIP
Proceedings of the 1997 International Workshop on Biophysics of Electron Transfer: Fundamental
Aspects and Applications, held October 8 ~ I 0, 1997, in Bressanone, ltaly
ISBN 978-1-4757-9518-9
© 1998 Springer Science+Business Media New York
Originally published by Plenum Press, New York in 1998
Softcover reprint of the bardeover 1st edition 1998
http://www. plenum.com
10987654321
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or Iransmitted in any form or by any
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permission from the Publisher
PREFACE
The present volume is a continuation ofthe EL.B.A. Forum Series which was initiated
in the spring of 1992 in Marciana Marina (Italy), with the first volume entitled From Neural
Networks and Biomolecu/ar Engineering to Bioelectronics published by Plenum Press in
1995.
Bioelectronics-miginally introduced in April, 1987, at a symposium hosted by
CIREF, a research consortium among leading high tech industries in Novara (Italy)---was
later defined in two successive consensus reports at the first (Bruxelles, 1991) and second
(Frankfurt, 1994) European Union Workshops on this widely interdisciplinary field, as "the
use ofbiological materials and biological architectures for information processing and sens
ing systems and devices down to molecular Ievel." lt is worth noting that these workshops
gave birth to the first European research program on "lnterfacing Biology with Electronics"
during 1996-1999, following the !arge Programma Nazionale Ricerca on "Technologies for
Bioelectronics" launched by the ltalian Ministry ofUniversities and Research in 1990.
In autumn, 1996, with the second volume, entitledMolecular Manufacturing, the em
phasis was placed on the ernerging parallel area of nanotechnology, independently initiated
in Palo Alto, Zurich, Genova, Mainz, and Tokyo by various groups (i.e., IBM, Xerox, Polo
Nazionale Bioelettronica, Max Planck Institutes), universities (i.e., Stanford, Genova, Rice,
Tokyo), and organizations (i.e., Foresight, Erato, Fondazione EL.B.A., Frontiers Research,
MITI) of different sizes, scopes, and latitudes.
The present third volume ofthe series highlights the various aspects ofthe biophysics
of electron transfer which has been ernerging as an independent branch of research. This vol
ume appears at a crucial moment, when significant progress is being made in this field, and
when technologies derived from it are being recognized as critical for the development ofbio
technology, electronics, and material sciences.
The significant roJe, ofthe Fondazione EL.B.A., the Istituto Cultura Trentina, and the
Istituto Nazionale Biostruttura e Biosistemi in the organization ofthis sixth course ofthe Na
tional School ofBiophysics in Bressanone, ltaly, October 8-10 1997, with the cooperation of
UNESCO and the ltalian Society ofBiophysics, are duly acknowledged as weil as forattract
ing and supporting (within the framework of Copernicus project number
ERBIC l5CT9608l 0 sponsored by the European Union) top Ievel scientists for the XIV EL.
B.A. Forum summarized in this volume and part ofthis course.
Polo Nazionale Bioelettronica must be acknowledged for bringing to light this series
and the enormous industrial potential of electron transfer in biopolymers.
I would like to express my gratitude particularly to Mr. Fabrizio Nozza and Andrea
Rossi ofthe Fondazione EL.B.A.
Claudio Nicolini
Member of the National Science and Technology Council
President ofthe Fondazione EL.B.A.
V
CONTENTS
Metalloprotein Engineering for New Materials, Drugs and Nanodevices ........... .
C. Nicolini
:n
Modulation of the Electron Transport System of Oxygenic Photosynthesis . . . . . . . . . .
G. Forti and G. Finazzi
Electron Transfer in Mitochondrial Steroid Hydroxylating Cytochrome P450
Systems: RoJe of Adrenodoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
R. Bernhardt
Preparation, Structural Characterization and Functional Coupling of Tethered
Membranes to Solid Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
W. Knoll, N. Bunjes, M. Denyer. C. Heibel, M. Matsuzawa, R. Naumann,
A. Offenhäusser, J. Rühe, E.-K. Schmidt, A. Sinner, and C. Sprößler
Targeted Expression of Mammalian Cytochromes P450scc and P4502b4 in Yeast
Saccharomvces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
M. A. Eldarov, V. E. Sidorovich, G. E. Pozmogova, and K. G. Skryabin
The Molecular Role of the Pufx Protein in Bacterial Photosynthetic Electron
Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I 03
F. Francia, P. Turina, B. A. Melandri, and G. Venturoli
Single Electron and Quantum Phenomena in Ultra Small Particles . . . . . . . . . . . . . . . . I 17
V. Erokhin, S. Carrara, and C. Nicolini
Electron Correlation in Quantum Molecular Biophysics: The Case Study of
Hemocyanin ....................................................... 139
P. Fariselli and R. Casadio
Electron Transfer Reactions in Multicopper Oxidases ........................... 161
L. Calabrese
The Optical Biosensor Study of Protein-Protein Interactions within Cytochromcs
P450 Containing Monooxygenase Systems ............................... 173
A. I. Archakov and Y. D. Ivanov
Index ................................................................. 195
VII
METALLOPROTEIN ENGINEERING FOR NEW MATERIALS, DRUGS AND
NANODEVICES
Claudio Nicolini
Istituto di Biofisica, Universita di Genova
Corso Europa 30, 16132 Genova Italy
Polo Nazianale Bioelettronica,
Via A. Moro 17, 57030 Marciana Marina (LI), Italy
Fondazione Elba
Via del Babuino 181, 00187 Roma Italy
INTRODUCTION
The purpose of this chapter is to provide an overview of present efforts on the engineering
thin films of of various metalloproteins, likewise P450 and C cytochromes and Photosynthetic
Reaction Center {RC), from ab initio considerations on the individual proteins in solution up to
the assembly and characterization of monolayers and multilayers. Over the years
metalloproteins, namely P450, C and Photosynthetic Reaction Centers, have become the
proteins of choice among the many currently under study in my laboratories towards the
implementation of drugs, materials and devices for numerous industrial applications. It should
be noted that several molecular manipulation techniques have been recently introduced which
could be utilized in order to optimize the properties of the above cytochromes in a wide range
of applications, namely:
self-assembly (Morgan et al., 1992; Hoffinann et al., 1992; Nicolini et al, 1995);
Langmuir-Blodgett/Langmuir-Shaeffer techniques and their modifications (Nicolini et al.,
1993; Antolini et al 1995; Nicolini, 1996b, 1997), including utilisation of reverse Iipid
micelies (Erokhin et al., 1994) and derivatization (Riccio et al, 1996) to form ordered thin
protein films;
site-directed chernical modifications complementing the above two techniques (Bernhardt et
al, this volume; Paskievitch et al., 1996).
The choice of metalloproteins as the most plausible materials to be used in bioelectronics is
determined by several of their inherent well-documented properties, of which the most
important is that the meta! sites present in those proteins are redox centers across which electron
transfer occurs along highly selective multidirectional pathways (Nicolini, 1996a); this is not the
case for low-molecular-weight electron carriers in inorganic materials such as silicon, that are
unidirectional and more uniformly (i.e., less selectively) reactive with respect to electron
Bwphysics of Electron Transfer and Molecu/ar Bwelectronics
Edited by C. Nicolini, Plenum Press, New York, 1998
transfer. These pathways are considered later in the chapter. Other considerations are as follows
for these classes of proteins:
metal sites are weil characterized by a number of spectral methods, so useful structural and
mechanistic information can be obtained using relatively inexpensive techniques;
• as a result of genetic engineering efforts, the genes for most of these systems are available
and can be expressed in large quantities in yeast or E. coli (Eldarov et al., 1996).
have well-studied biophysical, biochemical and enzymatic properties since they have served
as routine objects for many types of investigations;
have highly asymmetric distributions of electrostatic fields araund them. Same also have
asymmetric distribution of hydrophilic/hydrophobic amino acid residues over their surfaces.
Both of the two factors can be used for controlled association of these proteins into
supramolecular structures like thin films suitable for macroscopic characterization and/or
direct industrial applications.
from X-ray or NMR experiments, three-dimensional structures are known for most of them,
which are good starting points for designing or improving their properties, stabilities and
electron-transfer characteristics in several applications; for few others, likewise P450 sec and
P450 B24, modeling by homology might be the raute, until X ray crystallography and NMR
experiments still in progress will provide the needed informations.
METALLOPROTEINS SYSTEMS
Cytochrome C
Cytochrome Cis a small ["13 kDa, 104 amino-acids] soluble heme protein, which serves to
shuttle electrons between cytochrome reductase and cytochrome oxidase in the respiratory
electron-transfer chain. The high-resolution three dimensional structure of horse heart
Cytochrome C has been recently elucidated. The polypeptide chain is folded into a roughly
globular shape within which a heme packet is formed. The heme group, covalently linked to the
polypeptide chain via condensation of its vinyl peripheral substituents with cysteine in the 14 and
17 positions, is nearly completely buried within the surrounding protein matrix, but forms a
number of hydrogen bonds with nearby polar main-chain and side-chain groups and therefore
occupies a clearly hydrophilic local environment. Only four atoms of one edge of the
protoporphyrin IX heme prosthetic group are exposed to solvent (CMC, CAC, CBC, CMD
carbons), encircled by positively charged Arg and Lys. Two internally bound water molecules
seem to play apart in electron transfer. One internally bound water molecule (Watl25) mediates
a charged interaction between the heme propionate 01A and the guanidinium group of Arg-38.
The other water molecule (Watl12) is inside the protein molecule, centrally buried next to the
heme group.
Horse Cytochrome C has two surface tyrosines, in contact with the solvent, and two that
are buried. One of the latter, Tyr-67, is involved in H-bond donation to the sulphur atom of
Met-80 and a water molecule, which is in turn H-bonded to Thr-78. These are expected to be
relatively weak H-bonds. The other buried tyrosine, Tyr-48, donates and H-bond to a negatively
charged heme propionate group, which is expected to produce a strong interaction. The
pronounced evolutionary conservatism of aromaticity araund the heme group suggests that
these rings have an important roJe.
Photosynthetic Reaction Centre
Reaction Center from Rhodobactei. Sphaeroides. is a !arge (100 kDa) transmembrane
protein made of 3 subunits (L, M, and H) and is involved into the first stage of photosynthesis
2
providing photoinduced transmembrane electron transfer (I 00% quantum yield) in
photosynthetic bacteria (Branden and Tooze, 1991 ). Crystal structure of this protein has been
reported in the Iiterature (Allen et al., I987; Yeates et al., I987). Electron transfer chain formed
by a dimer ofBacteriochlorophyll (Bchl2), Bacteriopheophytin (Bphe), and 2 Quinones (Qa and
Qb) is shown in figure I along with the schematic subunit structure. In natural conditions Bchl2,
primary donor in the RC electron transfer chain, is reduced by a multiheme cytochrome attached
at the periplasmatic side of the protein; cytochrome is lost during the extraction from the
membrane (Branden and Tooze, 1991). The RC solution here used contains protein molecules
surrounded, in the hydrophobic region, by the detergent used for the extraction, i.e. spreading
solution is made oftwo components.
P450scc Cytochrome
There is no data on seif-exehange in cytochromes P450. This is related to the fact that is,
unlike other cytochromes, a complex enzymatic system involving other coenzymes (Adx and
AdR) in which the electron transfer must proceed in concert with other functions like substrate
binding and oxygen activation, and is performed with the help of cofactors - the iron-sulphur
protein, reductase, and, in some cases, cytochrome B5 (Porter and Coon, I99I ). Also, it follows
from its 3D structure (Figure I) that there is no way of bringing its possible inner electron
transfer pathways close to those of another P450 molecule unless its conformation dramatically
changes. Accordingly, the estimated seif-exehange rate for cytochrome P450 is nearly zero. The
structure of this protein has not yet been determined by X-ray crystallography, so sequence
homology modelling has to be applied to provide a model for this structure. Such a model has
been developed at this Institute, but a model is present also in the Protein Data Bank
(Vijayakumar and Salerno, I992). The key feature of cytochrome P450-catalysed reactions is
the transfer of electrons from NAD(P)H to either NADPH-cytochrome P-450 reductase in the
microsomal system (P450d) or a ferredoxin reductase and a non-haem iron protein (in the case
Figure I. Proposed model for the heme environment of the P450scc. The heme is attached to Cys423, and
its carboxyls interact with Arg357 and Arg421.
3
