Dimers of Azurin as model systems for electron transfer
Jongh, Thyra Estrid de
Citation
Jongh, T. E. de. (2006, September 12). Dimers of Azurin as model systems for electron
transfer. Retrieved from https://hdl.handle.net/1887/4554
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Introduction to electron transfer in a
biological context
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Abstract
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1.1 E lectron transfer in a biological context
The generation and subsequent transfer of electrons is a crucial component of a wide variety of biological processes. Once a flow of electrons is generated, for instance by light induced excitation of photoreceptive molecules, it will proceed along a series of acceptors and ultimately results in the formation of energy storage molecules such as ATP. In biology, electron acceptors or donors often are specific organic or inorganic cofactors incorporated into a protein scaffold. By encapsulating the redox centres in insulating protein matrices, nature assures that electrons are not passed on to wanton acceptors but are specifically and efficiently channelled along designated electron transfer (ET) pathways formed by a series of successive redox proteins. Because of the fact that the interacting centres are buried inside the proteins, electron transfer may have to proceed over distances as large as 20 Å, resulting in a relatively weak electronic coupling between the donor and acceptor sites. This long-range electron transfer (LRET) usually relies on electron tunnelling and is governed by the overlap between the wavefunctions of donor (D) and acceptor (A), which in turn is dependent on the DA distance separation as well as on the respective orbital orientations. As the tail of a wavefunction decays exponentially with distance, so does the electronic coupling strength (VD A). The rate of electron transfer (kE T) is directly related to the square of VD A according to Fermi’s rule:
Eq. 1
Here, FC denotes the Franck-Condon factor, a measure of the nuclear motion involved in the ET process. The classical theory for non-adiabatic electron transfer, pioneered in the early seventies by Levich and Dogonadze, allows this term to be approximated to the following expression, commonly referred to as the M arcus equation:[1]
Eq. 2
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parameter O is the energy that is required to reorganise the ligands and the solvent environment of the protein during ET, while 'Go is the standard free energy of
the reaction. As is apparent from equation 2, under conditions where -'G0 < O,
kETincreases with increasing driving force (-'Go) . This behaviour falls into what
is referred to as the ‘normal’ region. Somewhat counter-intuitively though, when -'G0 > O, the so-called 'inverted' region, a further increase of the driving force
results in a decrease of kET. The different situations are visualised in Figure 1.1. It is clear that fastest ET is achieved when -'G0 and O are perfectly matched so that the
activationless ET is purely under control of the electronic coupling. This parameter is strongly dependent on the intervening medium between the redox centres.
1.2. The protein m atrix
Although the actual uptake and donation of electrons is usually restricted to the organic or inorganic cofactors embedded in the protein, the protein matrix itself plays various essential roles in the ET process. As mentioned before, insulating the redox centres inside proteins helps to prevent spurious ET whilst determining the specificity and affinity with which appropriate electron acceptors are recognized by organizing the polypeptide chain into a 3-dimensional structure with electronic, Figure 1.1: Left: Potential energy diagram for ET between the reactants R resulting in the formation of products P, indicating the relationship between ET, -'G0 and
O. Right: Driving-force dependence of ET rates predicted by semi-classical theory (Eq. 2). Rates increase with driving force until they reach a maximum value (kETo) at
-'Go=O. Rates then decrease at higher driving forces (inverted effect). Figure made
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steric and/or hydrophobic determinants. Another important modulating function of the protein structure concerns the tuning of the redox potential of the reactive centres by adjusting the dielectric properties of the protein medium in the proximity of the redox centre. In this way, similar redox centres can be tuned to produce large differences in redox potentials. Furthermore, by shielding of the redox centre, outer-sphere solvent reorganization effects are greatly reduced. Additional reduction of the inner-sphere reorganization energy can be accomplished by stabilization of the geometry of the redox centre.[3] The lowering of reorganizational energy by
protein encapsulation is clearly illustrated in blue copper proteins, which effectively attenuate the large structural changes associated with reduction of copper complexes in aqueous solution.[4]
1.2.1. Intramolecular ET through protein media
The ways in which the protein matrix participates in mediating LRET has been a topic of interest for many years and several useful theoretical descriptions have arisen, the most widely employed of which will be discussed in more detail here. In electron tunnelling processes, kET is exponentially dependent on the efficiency with which different media are capable of mediating ET, expressed by the electronic distance decay constant E and the distance between donor and acceptor sites d (Eq. 3):
Eq. 3 Values for E can be as high as 3-5 Å-1 in vacuum and range from about 1.7 Å-1 in
water to as little as < 0.1 Å-1 for highly conductive phenylenevinylene molecular
wires [Figure 1.2].[5-7] For proteins, Dutton et al. have determined an averaged
decay factor of 1.4 Å-1 from a series of different protein/cofactor complexes, a
value approximately in between that of covalent systems and vacuum.[8] Although
the approach taken by these authors is often quoted as considering the protein a homogeneous ‘organic glass’-like medium with a single averaged electronic decay constant, it has since been refined to explicitly recognize that protein structure can play a modulating role in the form of an additional parameter termed the packing density U.[9] The empirically derived dependence of k
ET on the edge-to-edge DA
12 C h a p te r 1 Eq. 4 The distance parameter 3.6 Å in this equation represents the distance at van der W aals interaction at which kET,max§13 s-1. Even though the inclusion of a volume
occupancy parameter allows for some deviations from averaged behaviour, this uniform barrier (UB) model assumes that nature does not act by optimising specific ET pathways within a protein structure and does not attribute great importance to individual amino acid residues. In the approach developed by Beratan and co-workers on the other hand, ET is viewed as proceeding along a specific bridging pathway comprising three types of interatomic interactions: covalent bonds (C), hydrogen bonds (H) and through-space contacts (S), each with a distinct decay factor H.[10-12] The overall electronic coupling for a given tunnelling pathway (TP)
connecting the redox centres is taken as the product of its individual components:
where Eq. 5
V0 is the electronic coupling strength when the donor and acceptor sites would be in direct van der W aals contact. An important prediction from the TP model has been that strongly hydrogen bonded E-sheet structures form more effective mediators of long-range electronic coupling than D-helices do [Figure 1.2]. Based on the pathway analysis approach powerful search algorithms have been developed to identify electronically coupled pathways in known protein structures.[13;14]
These were shown to be very efficient in explaining why in certain ruthenated complexes of azurin differences in ET rate by orders of magnitude were found despite their similar DA distances.[15] More experimental support for the pathway
model has been provided by Farver et al. who observed LRET from the pulse radiolytically reduced intramolecular Cys3,26 disulfide bond to the copper centre in wild type and mutants of azurin (P. aeruginosa) and were thus able to identify distinct intramolecular pathways.[16-18] Although pathway calculations have proven
13 C h a p te r 1 1.2.2. Interprotein ET
All models discussed so far have been developed to treat mostly intraprotein ET and complex dynamics or solvent effects are of relatively little consequence. These factors will however become of major importance when considering intermolecular ET. One problem plaguing the identification of tunnelling pathways between protein complexes is that often the exact structure of the complex is not known or the complex is highly dynamic, not maintaining a single orientation for prolonged times, and the respective orientation of donor and acceptor is not clearly defined. Furthermore, true ET rates may become obscured when diffusional, gating or other regulatory effects become rate limiting. When no direct structural information on the complex is available, either from co-crystallization or from solution NMR studies, possible pathways can sometimes be identified by molecular dynamics simulations.[26] As
will be discussed in more detail in Chapter 2, chemical crosslinking can also be used as a means to render protein complexes amenable to structural analysis. In the pathway approach, intermolecular ET is treated by breaking down the pathway into two intramolecular steps, connecting the donor and acceptor sites to their respective protein edges, and one intermolecular through-space jump which may involve additional solvent interactions. The explicit involvement of solvent molecules in Figure 1.2: Tunneling timetable for ET through different media as a function of the edge-to-edge DA distance.[5] The
distance decay for ET through vacuum is shown as a black wedge, that for water as a grey wedge. The dashed lines illustrate the tunneling-pathway predictions for coupling along E-strands (E=1.0 Å-1) and
D-helices (E=1.3 Å-1) respectively, whilst
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mediating interprotein ET is gaining recognition. In this light, the electron self-exchange complex of wild type azurin, which reveals a water-based hydrogen bond network across the protein interface, is often mentioned.[27-29] Analysis of the ET
in crystals of zinc-doped cytochrome c, as well as in encounter complexes of the photosynthetic reaction centre with cytochrome c2 (R . sphaeroides) similarly suggests the involvement of water molecules in electronic coupling between redox centres. [30-32]
1.3. M olecular w ires
1.3.1. ET through molecular w ires
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Table 1.1: Dependence of different ET mechanisms on temperature and DA distance mechanism Temperature (T) DA distance (d) coherent tunnelling, ‘superexchange’ none exp(-Ed) incoherent, diffusive tunnelling none exp(-bd)
hopping exp(-a/T) d-1
* a and b denote constants independent of temperature or DA distance respectively (table after McC reery et al.[33])
1.3.2. DBA system properties
Molecular wires cover a broad spectrum of molecules and materials, including well-known examples such as carbon nanotubes, DNA and conjugated oligomers. Common oligomer building blocks include ethene, ethyne, thiophene, benzene, pyridine and aniline modules, all of which can be linked together to form extended conjugated chains [Figure 1.3].
Connecting multiple S-systems results in additional electron delocalization and increased conjugation, facilitating the ET process. Although the conventional linear representation of chemical structures may create the illusion of extensive conjugation, in reality the conjugation of a system may become severely distorted by geometric deviations from planarity. Functionalising the individual moieties by incorporating electron withdrawing or donating side-groups can also have a profound effect on the electronic bridge structure, allowing for a certain degree of tunability. It is clear that both geometric and electronic factors are important determinants of the effectiveness of molecular wires. Another important consideration in the design of DBA systems is the contact between the electrodes and the wire.[34] For
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can move freely into and out of the bridge, avoiding build up of charge at the interface. In general, strong chemical bonds lead to better electronic connection.
1.3.3. Applications of molecular wires in biosensors
The obvious appeal of molecular wires lies in the miniaturisation of electronic circuitry, eventually down to the single molecule level. Particularly interesting applications of molecular wires rest in their potential to effectively transmit protein mediated biosensory signals to a connected electrode or to drive enzymatic reactions in bionanotechnological devices.[35] By definition a biosensor is a biomacromolecule
based system able to detect the presence of a certain substrate and convert the stimulus into an electric signal which can then be relayed to an electrode. Enzymes are exquisitely suited for applications in biosensors due to their high natural sensitivity for specific compounds and their generally fast rate of catalytic turn-over. When binding and the subsequent conversion of a substrate leads to the generation of an electric current, a signal can be transduced to an electrode. However, as touched upon earlier, the protein shell itself is an essentially insulating medium that will impair efficient electronic communication. For relatively small proteins where the distance between the donor and acceptor sites does not exceed a25 Å, direct electrooxidation or reduction is still possible but larger molecules may be unable to directly interact with the electrode. The large glucose oxidase (GOx) molecule, which forms the basis of commercial glucose sensors, is a case in point. In order to enhance its efficiency as a biosensor, ferrocene derivatives are attached to the protein that act as electron relay stations.[36] These relay stations serve to reduce the
DA distance between subsequent redox centres and can be anchored to the protein surface by reaction with residues such as histidines or lysines.[37]
17 C h a p te r 1
This strategy has been put to the test in a number of enzyme/cofactor systems. Since the biomedically very interesting GOx enzyme is an FAD containing protein and these prosthetic groups can readily be extracted from the protein by acid precipitation whilst maintaining the native protein fold, reconstituted forms of GOx have been amongst the foremost to be investigated for their ability to transmit an electron flow to a connected electrode. Willner and co-workers employed amine functionalized FAD to tether apo-GOx to gold electrodes modified with a PQQ (pyrroloquinoline quinone) monolayer by treatment with the amine reactive agent EDC (1-ethyl-3-[(dimethylamino)propyl]-carbodiimide) so that the PQQ moiety serves as the mediator for the directional ET from the FAD to the electrode [Figure 1.4A].[48-50] The PQQ anchoring method has similarly been applied to reconstitute
apo-lactic dehydrogenase with modified NAD+ enabling the conversion of lactate
to pyruvate.[51;52]
Although the PQQ molecule exhibits substantial electron delocalization, the overall chain extending between the cofactor and the connected electrode is not strictly speaking considered a molecular wire. In fact, the usage of true molecular wires in conjunction with protein based biosensors is still in its infancy but is beginning to gather some momentum. Recently, Willner and co-workers have created a variation on their GOx sensor by substituting the PQQ relay with a highly conducting polyaniline based wire wrapped around a DNA template [Figure 1.4B].[53] It is to
18 C h a p te r 1
be anticipated that further exploits in this direction are not far behind as molecular wires may well represent the most efficient means for electron transduction in biosensors, provided that the wires can be connected to both the donor and acceptor sites with proper matching of their corresponding energy levels.
Although metal ion ligands are not normally viewed as cofactors, ligand substitution can have many of the same applications as cofactor reconstitution. The H117G mutant of azurin, which forms the basis of the work presented in Chapters 6 and 7, is a prime example. Mutagenesis of the copper ligand His117 for a much smaller glycine residue, creates a solvent exposed aperture in the protein which can then be filled by exogenous molecules such as imidazole or pyridine that ligate to the copper centre.[55-58] If these molecules are connected to a suitable wire structure,
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