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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Chapter
2
M odelling of interprotein electron
transfer by covalent and non-covalent
crosslinking
26 C h a p te r 2
Abstract
27 C h a p te r 2
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]
28 C h a p te r 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 Pseudom onas 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 E-sheets connected by an D-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
29 C h a p te r 2
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].
30 C h a p te r 2
2.1.2 E lectron 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 W T azurin, kese is on the order of 106 M-1s-1 and is only weakly dependent on pH
or ionic strength.[42] W hen 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 C h a p te r 2
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 C ovalent 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 C h a p te r 2
2.2.1 D irect crosslinking of azurin through the introduction of surface exposed cysteines
2.2.1.1 N 42C 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 1HJ2 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).
33 C h a p te r 2
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.
34 C h a p te r 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].
35 C h a p te r 2
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 K 27C 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-B M M E azurin
bis-36 C h a p te r 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 ( t 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 U 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 C h a p te r 2
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
38 C h a p te r 2
bifunctional linkers that are composed of cofactor or ligand-like moieties connected by a flexible spacer.
2.3.1 Dimerization of H 117G 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
39 C h a p te r 2
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 C h a p te r 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 C h a p te r 2
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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