Dimers of Azurin as model systems for electron transfer
Jongh, Thyra Estrid deCitation
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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Proefschrift
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden, op gezag van de Rector Magnificus Dr.D.D.Breimer,
hoogleraar in de faculteit der Wiskunde en Natuurwetenschappen en die der Geneeskunde,
volgens besluit van het College voor Promoties te verdedigen op dinsdag 12 september 2006
klokke 13.45 uur
door
Thyra Estrid de Jongh
geboren te Amersfoort in 1978
Promotiecommissie
Promotor: Prof. Dr. G.W. Canters Co-promotor: Dr. M. Ubbink
Referent: Prof. Dr. O. Farver (DFU Kopenhagen) Overige leden:
Prof. Dr. O. Einsle (Georg-August Universität Göttingen) Prof. Dr. M.T.M. Koper
Prof. Dr. J. Brouwer
Thyra de Jongh, Amsterdam 2006
Printed by: PrintPartners Ipskamp B.V., Enschede
Cover: Cartoon representation of a close-up of the interface between two molecules of wild type azurin (P. aeruginosa). The copper atoms are depicted as dark blue spheres. The sidechains of residues His117 are shown in stick representation. The ordered water molecules in the crystal lattice are indicated by semi-transparent blue spheres. (This picture was generated using PyMol 0.98).
Horizon
Een immer zinvol streven
Een doel dat nooit benaderd worden kon Mijn horizon
Een lijn zo recht en vast getrokken Geen enkel punt dat hem bepalen kon Mijn horizon
Zo vaak verborgen achter duist’re wolken Nooit een die hem verdrijven kon Mijn horizon
Altijd aanwezig en niet te bereiken Geen mens die hem begroeten kon Mijn horizon
Geen vaster punt dat steeds beweegt Geen beter doel dat steeds weer vlucht Ik ga er heen
Uit: Gedichten van een Kleine Zeeman op een Grote Zee Ricor de Jongh
Now, bring me that horizon!
Captain Jack Sparrow, Pirates of the Caribbean
Abbreviations
1,5-dip 1,5-di(imidazol-1-yl)pentane 1,6-dih 1,6-di(imidazol-1-yl)hexane azu azurin BMME bis-maleimidomethylether DA donor-acceptor pair DTT dithiotreitol ε extinction coefficientE. coli Escherichia coli
EPR electron paramagnetic resonance
e.s.e. electron self-exchange
ET electron transfer
HSQC heteronuclear single quantum correlation
IPTG isopropyl-b-thiogalactopyranoside
Kd dissociation constant
kese rate of electron self-exchange
kET rate of electron transfer
KPi potassium phosphate buffer
MES 2-morpholinoethanesulfonic acid
NMP N-methylpyrrolidone
NMR nuclear magnetic resonance
ox oxidised
P. aeruginosa Pseudomonas aeruginosa
red reduced
RT room temperature
TCEP tris(carboxyethyl)phosphine
UV-Vis ultraviolet and visible spectroscopy
WT wild type
Amino acids
A Ala Alanine M Met Methione
C Cys Cysteine N Asn Asparagine
D Asp Aspartate P Pro Proline
E Glu Glutamate Q Gln Glutamine
F Phe Phenylalanine R Arg Arginine
G Gly Glycine S Ser Serine
H His Histidine T Thr Threonine
I Ile Isoleucine V Val Valine
K Lys Lysine W Trp Tryptophan
Table of contents
Chapter 1:
Introduction to electron transfer in a biological context 7Chapter 2:
Modelling of interprotein electron transfer by covalent and non-covalentcrosslinking 25
Chapter 3:
Electron self-exchange in N42C-BMME azurin dimers as a function oftemperature 47
Chapter 4:
Electron transfer and complex formation in a pH sensitive dimer ofazurin
59
Chapter 5:
Electron transfer in a crosslinked protein dimer mediated by a hydrogen- bonded network across the interface 85Chapter 6:
Crystal structure of a non-covalent dimer of H117G azurin 111Chapter 7:
Hotwiring redox proteins by ligand reconstitution using conductingmolecular wires 131
Chapter 8:
General discussion, conclusions and perspectives 159Summary
170Samenvatting
174Appendices:
179Full colour images 180
Curriculum Vitæ 190
affiliations of the authors
Introduction to electron transfer in a
biological context
Chapter
1
Chapter 1
Abstract
Chapter 1
Introduction to electron transfer in a biological context
1.1 Electron 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 (VDA). The rate of electron transfer (kET) is
directly related to the square of VDA 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 Marcus equation: [1]
Eq. 2
10
Chapter 1
parameter λ 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 < λ, kET increases with increasing driving force (-∆Go) . This behaviour falls into what
is referred to as the ‘normal’ region. Somewhat counter-intuitively though, when -∆G0 > λ, 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 λ 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 matrix
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
λ. 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=λ. Rates then decrease at higher driving forces (inverted effect). Figure made
11
Chapter 1
Introduction to electron transfer in a biological context
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 b and the distance between donor and acceptor sites d (Eq. 3):
Eq. 3 Values for b 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 ρ.[9] The empirically derived dependence of k
ET on the edge-to-edge DA
12
Chapter 1
Eq. 4 The distance parameter 3.6 Å in this equation represents the distance at van der Waals interaction at which kET,max ≈ 1013 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 ε.[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 Waals contact. An important prediction from the TP model has been that strongly hydrogen bonded b-sheet structures form more effective mediators of long-range electronic coupling than α-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
Chapter 1
Introduction to electron transfer in a biological context
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 b-strands (b=1.0 Å-1) and
α-helices (b=1.3 Å-1) respectively, whilst
14
Chapter 1
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. Molecular wires
1.3.1. ET through molecular wires
15
Chapter 1
Introduction to electron transfer in a biological context
Table 1.1: Dependence of different ET mechanisms on temperature and DA distance
mechanism Temperature (T) DA distance (d)
coherent tunnelling, ‘superexchange’ none exp(-bd)
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 McCreery 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 π-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
metallic electrodes, wires are usually connected either by formation of a thiol bond, chemiadsorption onto the surface -potentially mediated by a self-assembled monolayer (SAM)- or hydrophobic interaction. In order to achieve optimal contact, a good orbital overlap between electrode and bridge is required such that electrons
Figure 1.3: Chemical groups that are frequently encountered as building blocks for
1
Chapter 1
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 ∼25 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
Chapter 1
Introduction to electron transfer in a biological context
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
Figure 1.4: GOx based sensors for the detection of glucose. The electrons generated
1
Chapter 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,
1
Chapter 1
Introduction to electron transfer in a biological context
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23
Chapter 1
24
Chapter
2
Modelling of interprotein electron
transfer by covalent and non-covalent
crosslinking
2
Chapter 2
Abstract
27
Chapter 2
Modelling of interprotein electron transfer by covalent and non-covalent crosslinking
Introduction
Many biological processes involve multiple partner proteins. The specificity as well as the affinity with which these form a complex is governed by the requirements to be met. In some cases, such as in antibody-antigen or signal transduction complexes, it will be necessary to form reasonably long lived and stable complexes. In contrast, there are many instances where rather than tight binding, a high rate of turnover is the main prerequisite and complexes will be formed with much lower binding affinities and significantly reduced lifetimes. Clear examples of the latter are found most notably amongst protein complexes involved in electron transfer (ET). The transport of electrons in cells proceeds along a chain comprised of multiple redox proteins, some of which may be membrane-bound, and shuttling of electrons between them is mediated by small soluble electron carrier proteins. The strong demand for fast and efficient ET, as well as the necessity for carrier proteins to be able to specifically interact with more than one partner protein, requires that these complexes are transient and their formation is governed by an interplay between the conflicting demands of sufficient specificity of recognition and that of rapid dissociation. Forces that help balance these needs include geometric surface compatibility, electrostatics or hydrophobic interactions.
To fully appreciate how exactly proteins interact, a detailed description of the surfaces involved in complex formation is needed. The study of ET protein complexes by conventional techniques such as X-ray diffraction is unfortunately hampered by their transient nature. To date very few of these short-lived and weakly binding complexes have been successfully co-crystallized. Most information on redox complex formation has come from NMR studies in which chemical shift perturbation mapping was used to identify protein binding sites and to specify the nature of the interaction.[1-5]
2
Chapter 2
cytochrome f and plastocyanin[9], cytochrome c and flavodoxin[10], cytochrome c
peroxidase and yeast cytochrome c[11] and several others. This chapter will focus
on the application of crosslinking in the formation of dimers of the redox protein azurin.
2.1. Azurin
2.1.1. Structure and function of azurin (P.aeruginosa)
Azurin is a small, 14 kDa, soluble redox protein belonging to the type I blue copper proteins, also known as cupredoxins. It is found in the periplasmic space of many gram negative bacteria including that of Pseudomonas aeruginosa, the source of the azurin discussed in this thesis. Its in vivo biological function has remained somewhat unclear, having been disproven as an obligatory partner of nitrite reductase (NIR) and as an alternative electron donor to cytochrome c551. It has been suggested that the physiological role of azurin is related to the cellular response to redox stress as increased azurin expression is observed under conditions of oxidative stress.[12] In vitro azurin is capable of interacting with nitrite reductase as well as with various
dehydrogenases.[13-17]
The overall structure of P. aeruginosa azurin consists of two b-sheets connected by an α-helix.[18] Buried about 7 Å beneath the protein surface, a single copper atom is
located that is coordinated by three strong ligands (His117, His46 and Cys112) that form a near-equatorial plane. On either side of the plane the ligation sphere of the copper atom is completed by a weak axial ligand (Gly45 and Met121). His117 is protruding towards the protein surface of an area known as the ‘northern face’ of the protein. Here it is surrounded by a cluster of hydrophobic residues, collectively referred to as the hydrophobic patch (HP) [Figure 2.1].
The presence of a hydrophobic region near the copper site is a conserved feature among all members of the cupredoxin family, although the exact size and amino acid composition of the region may vary.[19] The azurins are, however, unique in that
in all wild type crystal structures two molecules are found with their hydrophobic patches packed closely together [Figure 2.2].[18;20-22] As will be discussed in more
2
Chapter 2
Modelling of interprotein electron transfer by covalent and non-covalent crosslinking
within the HP have conclusively confirmed its involvement in the e.s.e. reaction of azurin. The introduction of both positive (M44K) or negative charges (M64E) in the HP was shown to lead to a marked pH dependent decrease of kese.[23-25]
In addition to its involvement in e.s.e., the HP has also been identified as the main site of interaction for ET between azurin and cytochrome c551, nitrite reductase and zinc cytochrome c.[24;26] Initially also a second site, located around histidine35, was
proposed as entry and/or exit point for electrons but mutagenesis studies in which the histidine residue was substituted for leucine, glutamine or phenylalanine have shown that this patch is not involved in self-exchange.[15]
Figure 2.1: Representation of P. aeruginosa
azurin seen from the northern face. Residues that make up the hydrophobic patch surrounding the copper ligand histidine117 (dark grey) are shaded and numbered. At the rim of the hydrophobic patch residue 42 (black) is situated. [Full colour image shown on page 180].
Figure 2.2: Cartoon depiction of
30
Chapter 2
2.1.2 Electron transfer and electron self-exchange in azurin
The relatively small size of azurin, combined with its distinctive spectroscopic properties, stability and high yield of expression have made azurin a classic model protein in the study of intramolecular and intermolecular biological ET. Much information on intramolecular long-range ET (LRET) has been obtained from studies on ruthenium-modified azurins in which excitable ruthenium complexes are anchored to residues on the protein surface.[27-32] Photochemical reduction of
the inorganic complex is succeeded by intramolecular ET to the Cu(II) centre. Alternatively, a unique intramolecular disulfide bond (Cys3-Cys26) that is located at the opposite end of the molecule with respect to the copper centre is pulse radiolytically reduced by generated CO2- radicals.[33-41] The free electron is then
intramolecularly transferred from the reduced RSSR- radical anion to the Cu(II)
ion. By mutagenesis of residues in the intervening protein medium, pathways for ET have been identified and the relative importance of specific residues could be determined. Azurin has also been used in a number of studies of intermolecular ET with various acceptors or donors.
A particularly attractive model system for the study of intermolecular ET in biological systems is the electron self-exchange reaction in which electrons are shuttled between the oxidized and reduced forms of a protein. Although this reaction is presumably non-physiological, its intrinsic simplicity has made it the focus of several studies centred on ET between redox proteins, including azurin. For WT azurin, kese is on the order of 106 M-1s-1 and is only weakly dependent on pH
or ionic strength.[42] When compared with e.s.e. rates found for other blue copper
proteins, that observed for azurin is relatively high.[43] This has been attributed to
31
Chapter 2
Modelling of interprotein electron transfer by covalent and non-covalent crosslinking
of a strongly coupled electronic pathway connecting the copper atoms.[44] Inspired
by the apparent agreement between the e.s.e. behaviour of azurin in solution and its distinctive protein packing observed in the crystal structure of azurin, several crosslinked dimers of azurin have been created to help further the understanding of some of the factors involved in interprotein ET.
2.2 Covalent crosslinking
The most commonly applicable method of covalent crosslinking of proteins is through use of bifunctional chemical crosslinking agents. The reactive groups targeted by such agents typically belong to either one of three distinct groups: 1) amine groups which can be recognized by agents such as glutaraldehyde or 1-ethyl-3-[(dimethylamino)propyl]carbodiimide (EDC), 2) thiol groups that are reactive towards, for instance, maleimides or iodoacetamides or 3) alcoholic groups which can be oxidized into more chemically reactive aldehyde groups by treatment with periodate. The natural abundance of alcoholic or amine containing amino acids, however, makes these less selective targets for crosslinking. In contrast, the low abundance of surface accessible thiols in most biomolecules combined with their high chemical reactivity, makes thiol groups ideal targets for controlled and selective chemical crosslinking. Unique sites for thiol based crosslinking can easily be introduced on a protein surface through site-directed mutagenesis. For azurin, a series of single and double cysteine mutants have been designed and expressed for various purposes [Table 2.1].
Table 2.1: Single and double cysteine mutants of azurin (P. aeruginosa)
Mutant Purpose
Q12C crosslinking with flavodoxinup, attachment of paramagnetic spin label (EPR)[45]
K27C Immobilization on gold surface (STM, AFM)[46], crosslinking[47], attachment of spin labels (EPR)[45]
N42C crosslinking[48;49], attachment of spin label s(EPR)[45], pulse radiolysis[50]
S118C crosslinking[47], attachment of spin labels (EPR)[45]
Q12C/K27C attachment of spin labels (EPR)mip
32
Chapter 2
2.2.1 Direct crosslinking of azurin through the introduction of surface exposed cysteines
2.2.1.1 N42C azurin
The relationship between the rate of interprotein ET and the structure of the association complex was explored by constructing covalent dimers of azurin that closely mimic the relative protein orientation of the proposed e.s.e. complex. Solvent exposed cysteines were engineered at position 42 on the protein surface enabling crosslinkage by intermolecular disulfide bond formation[48;49]. The crystal structure
of WT azurin shows Asn42 is located at the rim of the HP and formation of an intermolecular disulfide bond between cysteines at this position was expected to bring together the proteins with their hydrophobic patches facing each other and the copper centres positioned sufficiently close for fast e.s.e. [Figure 2.3, Figure 2.4].
Rates of e.s.e. were determined from NMR line broadening of the His46 proton resonance upon addition of small amounts of oxidized protein into a solution of reduced protein, as well as from simulation of the lineshape of the redox sensitive Val31 1Hγ2 resonance using the MEX/MEXICO software package.[51-53] Quite
unexpectedly, it was found that the disulfide crosslinked N42C azurin dimer displayed a very low kese within the dimer (keseintra < 10 s-1). In contrast, a high value
Figure 2.3: Proposed structure of the
e.s.e. complex of WT azurin based on the crystal packing orientation, showing the target site (N42) for covalent crosslinking (top right) by intermolecular disulfide bond formation between opposing monomers (bottom right).
Figure 2.4: Cartoon representation of
33
Chapter 2
Modelling of interprotein electron transfer by covalent and non-covalent crosslinking
for kese between dimers was determined (keseinter = 4.2 x 105 M-1 s-1), which is less
than an order of magnitude slower than for WT azurin. Elucidation of the crystal structure of the N42C disulfide bridged dimer offered a clear explanation for this initially surprising behaviour. It shows a dimer in which the two subunits are forced apart and the observed intramolecular Cu-to-Cu distance of 25.9 Å significantly exceeds the range of commonly observed donor-acceptor distances in biological systems with high ET rates [Figure 2.5].[54-56] The large distance was confirmed
in solution by pulsed electron-electron-double resonance (DEER) experiments indicating a distance of 26 Å.[57] Apparently, the introduced disulfide bond is too
short and rigid to allow the hydrophobic surfaces of the two monomers to come together to form a productive ET complex. The resulting ‘open’ structure, however, leaves the HP of one of the protein subunits exposed enough for interaction with a second dimer. The crystal structure shows two separate dimers in the asymmetric unit with an intermolecular Cu-to-Cu distance of 15 Å for the two nearest subunits, comparable to the Cu-to-Cu distance for WT azurin, which accounts for the high keseinter.
Figure 2.5: Cartoon depiction of the
34
Chapter 2
2.2.1.2 S118C azurin
An alternative site for direct crosslinking of azurin was created by replacement of a solvent exposed serine at position 118 by cysteine.[47] This residue is located close
to the HP and is adjacent to the copper coordinating ligand H117 [Figure 2.4]. Through formation of an intermolecular disulfide bond between the introduced cysteines, a short covalent path (H117-C118-C118-H117) connecting the copper centres is created [Figure 2.6].
As for the N42C disulfide linked dimer, the e.s.e. behaviour of S118C disulfide crosslinked azurin was studied by analysis of the redox sensitive Val31 proton resonance. Intriguingly, the S118C dimer was found to display anti-cooperativity in the two-step oxidation reaction of the dimer with a difference in redox potential between the first and second oxidation step of 33 mV, suggesting that the semi-reduced form of the dimer is stabilised over the fully oxidized form. Unlike for the N42C dimer, the position of the Val31 proton resonance of the semi-reduced form of the S118C dimer is slightly shifted compared to that of the fully reduced form suggestive of a small change in the geometry of the reduced copper centre upon oxidation of the opposing monomer. Together these observations imply that oxidation of the first copper centre induces strain in the molecule, disfavouring further oxidation. More indications for a structural rearrangement of the S118C dimer stem from the fact that, compared to other azurin dimers, several changes in resonance positions are detected and a number of NMR peaks are relatively
Figure 2.6: Schematic representation
35
Chapter 2
Modelling of interprotein electron transfer by covalent and non-covalent crosslinking
broadened. Finally, additional support for the presence of strain arises from the observation that the absorption maximum of the absorption around 630 nm is similarly dependent on the overall oxidation state of the dimer. The e.s.e. within, as well as between, dimers of S118C azurin was found to be very slow. Pathway calculations on the covalent path connecting the copper centres predict a keseintra
of approximately 60 s-1. From the experimental data, however, an upper limit of
only 3 s-1 was determined. This difference by more than an order of magnitude was
accounted for by assuming that the additional strain introduced by the disulfide bond has led to an increase in the reorganization energy of the copper site. The low keseinter is in turn explained in terms of a shielding of the HP as a result of
dimerization.
2.2.1.3 K27C azurin
To further explore the importance of properly orienting the association complex for efficient ET, dimers of azurin were created in which, rather than in a face-on orientation, the proteins were crosslinked at the “southern face” of the protein by replacement of Lys27 for a cysteine. Since position 27 is located at the far end of the molecule with respect to the copper centre, formation of a disulfide bond at this position is expected to result in the formation of a dimer in which the intramolecular Cu-to-Cu distance is maximized to about 60 Å [Figure 2.4]. Consistent with non-adiabatic ET theory, it was indeed found that the intramolecular e.s.e. in this complex is essentially absent (keseintra <10 s-1). The hydrophobic patches of each of the protein
monomers are, however, still accessible for interaction with other dimers, reflected by the fact that the experimentally observed keseinter is comparable to that of WT
azurin (2.6 x 106 M-1s-1) (unpublished results).
2.2.2 Covalent crosslinking through bifunctional thiol reactive spacers
The dimers formed by direct disulfide bond formation of N42C azurin, as well as those of S118C azurin, are examples of cases in which so-called ‘zero-length’ crosslinking leads to the formation of complexes that are not optimised for efficient ET. The use of molecular spacers that selectively react with groups on the protein surface may then help to alleviate the steric strain.
2.2.2.1 N42C-BMME azurin
bis-3
Chapter 2
maleimidomethylether (BMME) was introduced [Figure 2.8A].[49] Each
thiol-reactive moiety of this bifunctional linker was reacted with a single cysteine on the protein surface. The homodimer thus created was expected to have a higher degree of rotational flexibility around the site of crosslinking, thereby facilitating the formation of a productive intramolecular ET complex. It was found that the BMME crosslinked dimer of N42C azurin indeed displayed a high keseintra ( ≥ 5 x 104 s-1). The
crystal structure of the dimer confirmed the formation of an association complex in which the connected protein monomers are oriented in a fashion reminiscent of the WT crystal packing structure with a Cu-to-Cu distance of 14.6 Å [Figure 2.7]. The experimentally observed keseintra is in good agreement with the theoretically predicted
ET rate (kET = 4 x 105 s-1) for this donor-acceptor distance according to the empirical
model proposed by Dutton which describes kET as a function of the DA separation and the packing density ρ of the intervening medium [Eq. 1]:[56]
Eq. 1
In the dimer interface two hydrogen bonded water molecules were observed connecting to the copper coordinating H117 residues. As mentioned before, the presence of such ordered water molecules was first noted in the crystal packing of WT azurin. Through the formation of a hydrogen bond network these water molecules are thought to create a strong electronic coupling between the copper atoms. The importance of water molecules in the coupling between the redox centres is underscored by calculations of the electronic coupling element which predict a dramatic decrease of kese in the absence of this structurally ordered water.[44;58] Unlike
the direct disulfide crosslinked dimer of N42C azurin, the BMME crosslinked dimer displays a very low keseinter, which can be explained by the shielding of the HP in the
37
Chapter 2
Modelling of interprotein electron transfer by covalent and non-covalent crosslinking
2.3 Non-covalent crosslinking by cofactor or ligand reconstitution
An intriguing alternative to covalent crosslinking is presented by the fact that many proteins require non-protein based cofactors for their functioning. For many proteins their natural cofactor can be removed through chemical means and the resulting apo-protein can be reconstituted with a suitable artificial cofactor. By replacement of the native cofactor with a functionalized derivative, the spectroscopic and functional properties of a protein can be altered and non-native properties conferred onto the complex.
Hamachi and Hayashi have demonstrated some of the possibilities offered by this method by reconstitution of myoglobins with synthetic protohemin derivatives, thus creating proteins with artificial functionalities such as photoactivation, selective partner recognition, substrate sensing or membrane incorporation.[59-65] Of particular
interest is also the work of Willner et al. who utilised cofactor reconstitution of glucose oxidase and lactic dehydrogenase as a means of establishing electrical contact between the enzyme and an electrode. Furthermore, cofactor reconstitution enables highly specific non-covalent crosslinking of proteins through use of
Figure 2.7: Crystal structure of the N42C-BMME
3
Chapter 2
bifunctional linkers that are composed of cofactor or ligand-like moieties connected by a flexible spacer.
2.3.1 Dimerization of H117G azurin
The principle of ligand reconstitution has been extended to azurin. It has been shown that it is possible to replace one of the copper ligating histidines, H117, by a glycine without severely disrupting the overall protein structure.[66;67] The removal
of the coordinating imidazole side chain leaves a gap in the coordination sphere of the copper ion that, since H117 normally protrudes towards the protein surface, can be filled by exogenous copper coordinating ligands.[67] Depending on the nature of
the external ligand, the spectroscopic features of the mutant protein resemble either that of the native Type-1 site or that of a Type-2 copper site. In the absence of any external ligands, the coordination sphere of the copper ion is completed by water, resulting in the formation of a Type-2 like copper site.[68] Addition of monodentate
ligands like Cl-, Br-, imidazole (derivatives) or pyridine (derivatives) was shown to
restore the spectroscopic properties of the native Type-1 site.[69;70] Reconstitution
with derivatives of imidazole, an analogue of the natural histidine, allows direct ‘hotwiring’ of the copper site.
Van Pouderoyen et al. have demonstrated how hotwiring can be used to achieve non-covalent crosslinking of azurin.[71;72] Bifunctional molecules were synthesized
in which two imidazole moieties are connected by a flexible alkane chain, the length of which was varied [Figure 2.8B-D]. It was found that addition of these
bis-imidazolyl(CH2)n linkers with either n=5 or n=6 led to dimerization of
Cu(II)-Figure 2.8: Bifunctional linkers
3
Chapter 2
Modelling of interprotein electron transfer by covalent and non-covalent crosslinking
H117G azurin and to restoration of the spectroscopic WT features. In contrast, the linker with n=4 proved to be too short for efficient dimer formation. EPR spectra of H117G azurin dimerized with 1,5-di(imidazol-1-yl)pentane or 1,6-di(imidazol-1-yl)hexane respectively revealed small, yet distinct differences in the geometry of the copper site. The H117G azurin-1,5-di(imidazol-1-yl)pentane complex exhibited a spectrum composed of two slightly different Type-1 copper sites. Most likely, the linker is unable to bind to two proteins in exactly the same fashion as a result of steric constraints and the two imidazole rings are forced into slightly different orientations with respect to the copper sites. For the somewhat longer 1,6-di(imidazol-1-yl)hexane linker this steric constraint was relieved and the EPR spectrum showed only a single Type-1 species. The work presented in Chapter 6 discusses a crystal structure of Zn-H117G azurin dimerized with 1,6-di(imidazol-1-yl)hexane, the first crystal structure of a metal-bound and ligand-coordinated form of H117G azurin. Such structural information on crosslinked or reconstituted systems is of vital importance for their potential application in biotechnological devices such as those discussed in Chapter 1.
Conclusions and prospects
40
Chapter 2
The various examples presented in this review stress that care must be taken in the design of efficient crosslinked ET complexes. It has long been thought that the use of short crosslinkers is preferable in the study of redox complexes in order to minimize the distance between the respective redox centres. Often, however, this strategy has resulted in the formation of complexes that proved to be inefficient. The structure and e.s.e. properties of the disulfide crosslinked dimer of N42C azurin illustrate how short linkers can impede the formation of properly oriented ET complexes by restricting the freedom of motion of the partner proteins. Zero-distance crosslinking may even cause a slight distortion of the redox centre itself, as demonstrated by the redox behaviour of S118C azurin. These findings clearly show how even relatively small changes in energy or geometry can have a profound effect on the rate of ET. In such cases, the use of longer and more flexible spacers can relieve the steric constraints and allow the formation of more favourable complexes that exhibit fast ET. The N42C-BMME azurin dimer serves as a good example of this principle. The crystal structures of both wild type and N42C-BMME azurin reveal the presence of ordered water molecules in the protein interface. The formation of a hydrogen bond network connecting the copper centres may be of vital improtance in the efficient electronic coupling of the redox centres as shown by pathway calculations of the azurin dimer that highlight that solvent molecules should be explicitly taken into account when analyzing ET pathways in redox complexes.
41
Chapter 2
Modelling of interprotein electron transfer by covalent and non-covalent crosslinking
of the H117G-imidazole complex is possible, provided that the rate of reoxidation is faster than the rate of dissociation of imidazole.[73] In principle this implies that,
as long as linkers with sufficiently strong electronic coupling are used, the formation of redox active complexes of H117G azurin is possible.
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45
Chapter 2
Modelling of interprotein electron transfer by covalent and non-covalent crosslinking
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4
Chapter
3
Electron self-exchange in N42C-BMME
azurin dimers as a function of
4
Chapter 3
Abstract
The electron self-exchange (e.s.e.) reaction in a covalent homodimer of N42C-BMME azurin was investigated as a function of temperature in an attempt to determine the reorganization energy λ. Rates of intramolecular e.s.e. were obtained by simulation of the redox sensitive Val31 1Hγ2 resonance in NMR spectra recorded
at temperatures between 312 and 272K. At T ≥ 303K a lower limit for keseintra was
found of ≥ 2x104 s-1 which is consistent with previous reports. At lower temperatures
