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Protein Binding and the Electronic Properties of Iron(II) Complexes: An

Electrochemical and Optical Investigation of Outer Sphere Effects

Kylie D. Barker,‡ Amanda L. Eckermann,‡ Matthew H. Sazinsky,‡,⊥ Matthew R. Hartings,‡ Carnie Abajian,‡

Dimitra Georganopoulou,† Mark A. Ratner,‡ Amy C. Rosenzweig,‡,§ and Thomas J. Meade*,‡,§,|

Departments of Chemistry, Biochemistry and Molecular and Cell Biology, Neurobiology and Physiology, and Radiology,Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208. Received June 17, 2009; Revised Manuscript ReceivedSeptember 1, 2009

Metalloenzymes and electron transfer proteins influence the electrochemical properties of metal cofactors bycontrolling the second-sphere environment of the protein active site. Properties that tune this environment includethe dielectric constant, templated charge structure, van der Waals interactions, and hydrogen bonds. Bysystematically varying the binding of a redox-active ligand with a protein, we can evaluate how these noncovalentinteractions alter the electronic structure of the bound metal complex. For this study, we employ thewell-characterized avidin-biotin conjugate as the protein-ligand system, and have synthesized solvatochromicbiotinylated and desthiobiotinylated iron(II) bipyridine tetracyano complexes ([Fe(BMB)(CN)4]2- (1) and[Fe(DMB)(CN)4]2- (2)). The binding affinities of 1 and 2 with avidin are 3.5 × 107 M-1 and 1.5 × 106 M-1,respectively. The redox potentials of 1 and 2 (333 mV and 330 mV) shift to 193 mV and 203 mV vs Ag/AgClwhen the complex is bound to avidin and adsorbed to a monolayer-coated gold electrode. Upon binding to avidin,

the MLCT1 band red-shifts 20 nm for 1 and 10 nm for 2. Similarly, the MLCT2 band for 1 red-shifts 7 nm andthe band for 2 red-shifts 6 nm. For comparison, the electronic properties of 1 and 2 were investigated in organicsolvents, and similar shifts in the MLCT bands and redox potentials were observed. An X-ray crystal structureof 1 bound to avidin was obtained, and molecular dynamics simulations were performed to analyze the proteinenvironment of the protein-bound transition metal complexes. Our studies demonstrate that changes in the bindingaffinity of a ligand-receptor pair influence the outer-sphere coordination of the ligand, which in turn affects theelectronic properties of the bound complex.

INTRODUCTIONThe properties of coordinatively saturated transition metal

complexescanbeprofoundlyinfluencedbytheirenvironment(1-7 ).The ligands of a transition metal complex can interact withsurrounding solvent molecules, macromolecular species, orproteins to affect the electronic state of the metal complex. Thesenoncovalent interactions (known as outer-sphere or second-sphere coordination) include hydrogen bonding, π -π stacking,electrostatic, hydrophobic, and van der Waas interactions (8-11).

The concept of outer-sphere coordination was introduced byWerner to explain phenomena such as solvents of crystallizationand the formation of adducts between amines and coordinativelysaturated complexes (12, 13). In 1981, Stoddart and co-workerspresented the first crystallographic evidence of this type of interaction, in which a crown ether is associated with aPt-ammine complex through noncovalent interactions (14). Inrecent years, it has been shown that interactions with the secondsphere of coordination complexes affect the physical properties.For example, interaction of 18-crown-6-ether with the secondsphere of a series of ruthenium-ammine complexes results inchanges in the spectroscopic and electrochemical properties of the complexes (6, 7 ).

Second-sphere coordination is important in biological pro-cesses such as iron siderophore recognition, and is currently

being investigated as a means to mimic the diverse functionalityof metalloenzymes (9, 15, 16 ). The ability to modulate theproperties of transition metal complexes through second-sphereligand design is of interest for the development of artificial

metalloenzymes that combine the features of both heterogeneousand enzyme catalysis (17, 18). Ward and co-workers usechemogenetic protein engineering to optimize artificial metal-loenzymes comprising a catalyst anchored to a biologicalmacromolecule (19-21). Second-sphere coordination is knownto play an important role in activity and enantioselectivity inthese catalytic reactions (17, 19-22).

Previously, we reported a series of redox active transitionmetal-modified biotin complexes designed to probe the effectsof protein binding on the electrochemical response (Figure 1)(23). The biotin-avidin system was chosen as a model due toits strong binding interaction (K a ) 1015 M-1) and extensivestructural characterization (24-26 ). Electrochemical studiesdemonstrated that the avidin-bound complexes are shielded andunable to effectively interact with the electrode. However, thebound complexes can be accessed via electrochemical mediators,suggesting that the complex is only partially shielded in thebinding pocket of the protein (23). Quantitative results couldnot be obtained for the electrochemical potential of the protein-bound complex. Therefore, to investigate the effects of theprotein binding on the electronic properties of the boundcomplex, we chose to use [Fe(bpy)(CN)4]2- complexes (Figure1). [Fe(bpy)(CN)4]2- complexes possess electrochemical andoptical properties that are highly sensitive to the localenvironment (4, 27 -29).

To investigate how differences in ligand-receptor bindingenergetics influence the second-sphere coordination environment

* E-mail: [email protected].‡ Department of Chemistry.⊥ Current Address: Department of Chemistry, Pomona College.† Ohmx Corporation, 1801 Maple Avenue, Suite 6143, Evanston,

Illinois 60201.§ Departments of Biochemistry and Molecular and Cell Biology.| Departments of Neurobiology and Physiology and Radiology.

Bioconjugate Chem. 2009, 20, 1930–1939 1930

10.1021/bc900270a CCC: $40.75 2009 American Chemical Society Published on Web 09/29/2009



of a small molecule when bound to a protein, we designed tworedox-active, solvatochromic transition metal complexes that

bind to avidin with different affinities. Herein, we directlycorrelate systematic modifications of the complex environmentto observed changes in electrochemical and spectroscopicsignals. The influence of avidin binding on the electrochemicaland spectroscopic properties of [Fe(BMB)(CN)4]2- (1) and[Fe(DMB)(CN)4]2- (2) is discussed in light of X-ray crystal-lographic, electrochemical, optical, computational, and thermo-dynamic studies.

EXPERIMENTAL SECTION

Materials and Methods. Deglycosylated avidin was usedfor crystallization studies and obtained from Belovo, Inc. Eggwhite avidin, used for thermodynamic studies, was obtainedfrom Molecular Probes. Poly(ethylene glycol) was obtained fromFisher Scientific. All other chemicals were obtained fromAldrich. K2[Fe(BMB)(CN)4] (1) and K2[Fe(DMB)(CN)4] (2)were prepared according to previously reported methods (23).

Electrochemistry. Cyclic voltammetry experiments werecarried out in a conventional three-electrode cell using a CHInstruments 660A workstation (Austin, TX), glassy carbonworking electrode (3 mm dia, CH Instruments), Pt wire counterelectrode, and Ag/AgCl reference electrode. The reference electrodewas calibrated to Fc/Fc+. The electrode was polished with aluminaon a polishing cloth and the sample purged with N2 before eachexperiment. All electrochemical potentials ( E 1/2) are reported vsAg/AgCl [where E 1/2 is defined as ( E pa + E pc)/2].

Cyclic voltammograms of 1 were collected in solvents of varying acceptor number (AN). A 200 µL aliquot of a 50 mMaqueous solution of 1 was added to 3 mL of a 50/50 solvent/ 100 mM PBS buffer (pH 7.2) mixture where the solvents werewater (AN ) 55), methanol (AN ) 41), DMSO (AN ) 19), orDMF (AN ) 16) (30). Cyclic voltammograms of 1 and 2 in0%, 10%, 20%, 30%, 40%, and 50% DMF in PBS buffer werecollected. Samples were prepared by adding appropriate amountsof solvent and water to 1.5 mL 100 mM PBS buffer (pH 7.2)to make a 3 mL sample with a constant electrolyte concentrationof 50 mM. A 200 µL aliquot of a 50 mM aqueous solution of 1 or 2 was added to the solution. For example, the 10% DMFsample was prepared by adding 0.3 mL DMF and 1.2 mL waterto 1.5 mL 100 mM PBS buffer.

In order to obtain cyclic voltammograms of 1 and 2 boundto avidin, a gold ball electrode was prepared by melting theend of a 0.127-mm-diameter gold wire into a ball approximately0.75 mm in diameter. The electrode was cleaned electrochemi-

cally by immersing it in 2 N H2SO4 and cycling the potentialbetween 0 and 1.6 V at a scan rate of 500 mV/s. The electrodewas rinsed with water followed by ethanol and immediatelyimmersed in a 5 mM ethanolic solution of 11-mercaptounde-canoic acid for 16 h. The electrode was washed with ethanolfollowed by water and 10% NH4OH(aq). It was then immersedin an aqueous solution consisting of 0.1 mM 1 or 2 and 4 mg/ mL avidin for 2 h. The electrode was washed with water, andcyclic voltammograms were obtained in 100 mM PBS buffer(pH 7.2).

Absorption Spectroscopy. UV-vis absorption studies wereperformed using an Agilent 8453 spectrophotometer (FosterCity, CA). Samples were prepared as described for electro-chemical experiments. UV-vis spectra of 1 and 2 bound toavidin were obtained by preparing a solution of each in 100mM PBS buffer and adding a 1.5-fold excess of avidin.

Isothermal Titration Calorimetry. Thermodynamic studiesof 1 and 2 were carried out using a VP-ITC microcalorimeterfrom MicroCal, Inc. (Northampton, MA). In a typical experi-ment, the reference cell was filled with pure distilled water. Thereaction cell was filled with 1.8 mL of a 6 µM solution of avidin(24 µM avidin protomer) in pH 7.0 phosphate buffer. Theinjection syringe was filled with a 300 µM solution of 1. After

thermal equilibration to 25 °C, 1 was injected into the reactioncell in 5 µL aliquots every 200 s until saturation was achieved(18 injections). A reference offset of 25% was used. Theexperiment was repeated injecting 1 into pH 7.0 phosphatebuffer. The data were subtracted from the binding data to correctfor heats of dilution. The data were fit using the Origin 7.0OneSites model to determine binding constants (K a) and heatsof binding (∆ H ). The above procedure was repeated forcompound 2.

Crystallization and Data Collection. The K2[Fe(BMB)-(CN)4] complex was incubated with avidin for 1 h at roomtemperature. The avidin-[Fe(BMB)(CN)4]2- complex was crys-tallized at 18 °C by using the sitting drop vapor diffusionmethod. A protein solution containing 3.0 mg/mL avidin boundby [Fe(BMB)(CN)4]2- in phosphate buffer pH 7.0 was combinedwith an equal volume of precipitant solution comprising 100mM sodium acetate (pH 4.6) and 25% PEG 3000. The resultingcrystals were harvested after six weeks. The crystals werehexagonal, space group P3221, with unit cell dimensions a ) b

) 74.20, c ) 85.82.X-ray data were collected at 100 K at the Advanced Photon

Source on beamline 5-ID from crystals flash-frozen in liquidnitrogen after soaking in a cryo-protectant composed of the wellsolution plus 20% ethylene glycol. The diffraction data wereintegrated with MOSFLM (31) and scaled by using SCALA(32). The statistics are presented in Table 5.

Structure Determination and Refinement. Phases were

determined by molecular replacement using the programPHASER (33) and avidin from the pdb file 1LDO as the searchmodel. The ligand-bound form of avidin was modeled withXtalView (34), refined with CNS (35), and validated withPROCHECK (36 ).

Molecular Modeling. Force field parameters were derivedfor [Fe(BMB)(CN)4]2- and [Fe(BMB)(CN)4]2-. Atomic partialcharges were taken from the Mulliken population analysis of ab initio geometry minimizations. These calculations werecarried out at the Hartree-Fock (HF) level in the programGamess (37 ). A Hay-Watt effective core potential was usedto describe the iron atoms (38). The carbon and nitrogen atomsin the bipyridyl and cyano ligands were described by 6-31+G*basis set (39). All other atoms were described by the 6-31 G*basis set (40). Geometries and charges were calculated for both2+ and 3+ metal ionization states.

Figure 1. Structures of [Fe(BMB)(CN)4]2- and [Fe(DMB)(CN)4]2-.Solvatochromic [Fe(bpy)(CN)4]2- complexes are targeted to bind avidinby modification with biotin (1) or desthiobiotin (2) through amidederivatization of 4-aminomethyl-methyl-2,2′-bipyridine.

Outer Sphere Effects of Iron(II) Complexes Bioconjugate Chem., Vol. 20, No. 10, 2009 1931



Iron-ligand bonds were modeled using the points-on-a-sphere

methodology (41). In this method, iron-nitrogen and iron-carbonbond lengths are held constant (acquired from crystal structuresof similar compounds) (42) and the atom-iron-atom bondangles are determined by the electrostatic interaction of theatoms around the metal center.

The crystal structure of the biotin-avidin complex (obtainedfrom the Protein Data Bank) was used as the starting geometryfor the molecular dynamics simulation (26 ). Hydrogen atomsand other atoms missing from the crystal structure were addedreferencing standard amino acid residues from AMBER. Theab initio optimized geometry coordinates for the [Fe(bpy)-(CN)4]2- complex were appended to the coordinates of thecrystal structure of avidin bound biotin for each of the fourbinding avidin protomers. The energy of the avidin/{[Fe(BMB)-

(CN)4]2-

}4 tetrameric complex was minimized using a combinedsteepest descent and conjugate gradient minimization technique.The minimized structure was solvated with a 75 Å × 75 Å ×

75 Å box of water of approximately 13 800 TIP3P watermolecules. The solvated protein structure was minimized usingthe method described above.

Molecular dynamics (MD) simulations were carried out forthe avidin/{[Fe(BMB)(CN)4]2-}4 tetrameric complex using the

Amber 94 force field (43). Constant volume periodic boundaryconditions were employed; the simulation boundary box con-structed was slightly larger than the extent of the water bufferused to solvate the protein. A dielectric constant of 4 wasassigned to the boundary box to dampen the effects of electrostatics on the system. MD simulations were run for 1 ns

recording energy and trajectory information every picosecond.The isopotential surfaces were rendered with HARLEM froma calculation of the electrostatic potential by solving thePoisson-Boltzmann equation using the method of finite dif-ferences (44). Electrostatic calculations were carried out usingthe partial charges on the avidin/{[Fe(BMB)(CN)4]2-}4 complexto represent the charge distribution. The static dielectric constantas a function of position, also used in the Poisson equation,was mapped by defining the low dielectric volume of the proteinby a solvent-accessible surface (SAS). A solvent probe radiusof 1.4 Å was used to define the SAS. Atoms contained insidethe boundary of the surface are assigned a low dielectric constantof 4, outside the surface the dielectric is assigned value of 80for water. These calculations yield the isopotential surface

surrounding the modified ligands. Calculations were made foratoms within a 10 Å shell around the binding ligand. Thiscalculation provides an estimate of the electric field immediatelysurrounding the ligand and indicates portions of the protein withnet positive or net negative charge.

RESULTS

Electrochemistry. The E 1/2 of 1 and 2 in PBS pH 7.2 are333 mV and 330 mV, respectively. These values are inagreement with previously reported values and comparable tothatof[Fe(4,4′-dimethyl-2,2′-bipyridine)(CN)4]2-(312mV)(23,45).The E 1/2 of 1 increases with increasing solvent acceptor number(AN) where AN describes the Lewis acidity of the solvent(Table 1, Figure 2) (30, 46 ). When DMF is introducedincrementally to an aqueous solution of 1 or 2, the E 1/2 decreaseslinearly (Tables 2 and 3, Figure 3). The plot of current (i) vs

square root of scan rate (V) is linear for 1 and 2 in all solventsystems. The average ∆ E p (difference of the peak potentials)for 1 and 2 is 64 mV and is consistent for all solvent systems.The ia / ic ratio is 0.8 for 1 and 1.0 for 2 in all solvent systems.

An electrochemical signal is not observed for 1 or 2 boundto avidin in solution (23). When adsorbed to a mercaptounde-canoic acid (MUA) SAM-coated gold electrode, 1 has an E 1/2

of 193 mV and 2 has an E 1/2 of 203 mV. At 100 mV/s, the ∆ E pfor 1 in this experiment is 85 mV with an ia / ic ratio of 1.1(Supporting Information Figure S1). The ∆ E p for 2 is 75 mVwith an ia / ic ratio of 1.1 (Figure 4).

Absorption Spectroscopy. For an aqueous solution of 1, acharge transfer (CT) band is observed at 293 nm and two MLCTbands are observed at 341 nm [metal dπ f bipyridine π * (2)(MLCT2)] and 480 nm (metal dπ f bipyridine π * (1)(MLCT1)) (47 ). The λmax of the MLCT peaks are red-shifted

Table 1. Variation of MLCT Wavelengths and ElectrochemicalPotential of K2[Fe(BMB)(CN)4] with Acceptor Number (AN)

solvent AN MLCT1 (nm) MLCT2 (nm) E 1/2 (mV)

water 55 480 341 333MeOH 41 496 348 257DMSO 19 512 356 195DMF 16 519 357 181avidin - 500 348 193

Figure 2. (A) Correlation of the K2[Fe(BMB)(CN)4] MLCT bandswith solvent acceptor number. Closed (•) symbols represent MLCT2;open symbols (O) represent the MLCT1. (B) Correlation of theK2[Fe(BMB)(CN)4] E 1/2 with solvent acceptor number. Cyclicvoltammograms were collected at 100 mV/s using a glassy carbonelectrode and Ag/AgCl reference. Values are reported vs Ag/AgCl.

Table 2. Variation of MLCT Wavelengths and ElectrochemicalPotential of K2[Fe(BMB)(CN)4] with Increasing DMF

% DMF MLCT1 (nm) MLCT2 (nm) E 1/2 (mV)

0 480 341 33310 490 345 30120 496 347 27030 510 349 246

40 514 352 22150 519 357 211avidin 500 348 193

Table 3. Variation of MLCT Wavelengths and ElectrochemicalPotential of K2[Fe(DMB)(CN)4] with Increasing DMF

% DMF MLCT1 (nm) MLCT2 (nm) E 1/2 (mV)

0 486 345 33010 494 351 30220 501 357 26930 510 360 23840 519 363 21250 529 368 184avidin 496 351 203

1932 Bioconjugate Chem., Vol. 20, No. 10, 2009 Barker et al.



linearly with decreasing solvent acceptor number (Table 1,Figure 2). When DMF is introduced incrementally to an aqueoussolution of 1, the λmax values of the MLCT peaks are red-shiftedproportionally (Table 2, Figure 5). Upon addition of avidin to1 in PBS buffer, the MLCT1 band shifts 20 nm (833 cm-1)and the MLCT2 band shifts 7 nm (590 cm-1) (Table 2). Whenan excess of biotin is added to the solution of 1-avidin, the λmax

values of the MLCT peaks are shifted back to the originalvalues.

For an aqueous solution of 2, a CT band is observed at 293nm and two MLCT bands are observed at 345 nm (MLCT2)and 486 nm (MLCT1). When DMF is introduced incrementallyto an aqueous solution of 2, the λmax values of the MLCT peaksare red-shifted proportionally (Table 3, Figure 5). Upon additionof avidin to 2 in buffer, the MLCT1 band shifts 10 nm (415

cm-1) and the MLCT2 band shifts 6 nm (495 cm-1) (Table 3).When an excess of biotin is added to the solution of 2-avidin,the λmax values of the MLCT peaks are shifted back to theoriginal values.

Isothermal Titration Calorimetry. For 1 and 2, the amountof energy released per mole of complex titrated was plottedversus the molar ratio of complex to protein. The curve wasbest fit using the Origin OneSites model to give the bindingconstant (Supporting Information Figure S1). The reduced χ2

value was 1.3 and 1.1 for 1 and 2, respectively. Bindingconstants and heats of binding for 1 and 2 binding to avidinare summarized in Table 4.

Structure of the [Fe(BMB)(CN)4]2-

-Avidin Complex. Com-plex 1 bound to both protomers in the 3.1 Å resolution crystalstructure of the avidin complex is shown in Figure 6. Asexpected, the biotin group binds to a site identical to onepreviously characterized (26 ). In one protomer, electron densityfor the [Fe(BMB)(CN)4]2- group is observed on the surface of the protein, sandwiched among three loops: L3,4; L5,6; and L7,8(Figure 6B). This configuration appears stabilized by severalhydrogen bonding pairs including interactions of N12, S16, Y33,T35, T77, and N118 with the ureido portion of the complex,S73 and S75 with the carbonyl of the amide bond, and S31 andS102 with the transition metal portion of the complex. In thesecond protomer, electron density for the [Fe(BMB)(CN)4]2-

group and residues 41-44 on the L3,4 loop were not observed.

Molecular Modeling. In the course of the molecular dynam-ics simulation, several conformations were found to be acces-sible to 1 when bound to avidin (Supporting Information FigureS2). The conformation in closest agreement with the crystalstructure was chosen to compare the second-sphere environmentof 1 and 2 when bound to avidin. In this conformation, thedistances from the R carbon of Ser 41 and the metal atom of 1

and 2 are 5.76 and 5.92 Å, respectively. The distance betweenSer 101 and the metal atom was found to be approximately 10Å for both 1 and 2. The distance between Ser 102 and the metalatom was found to be approximately 5 Å for both 1 and 2. Lossof β architecture in strands 3 and 4 is observed and loops 7,8and 5,6 are disordered for 2 bound to avidin. The models of 1

and 2 bound to avidin are compared in Figure 7. The isopotentialsurface of the portion of the protein that surrounds avidin (Figure

Figure 3. Electrochemistry of K2[Fe(BMB)(CN)4] and K2[Fe(DMB)(CN)4] in increasing concentrations of DMF in PBS buffer at pH 7.2. (A)Representative cyclic voltammograms of K2[Fe(DMB)(CN)4] (B) Correlation of the E 1/2 of K2[Fe(BMB)(CN)4] with increasing amounts of DMF(C) Correlation of the E 1/2 of K2[Fe(DMB)(CN)4] with increasing amounts of DMF. Cyclic voltammograms were collected at 100 mV/s using aglassy carbon electrode and Ag/AgCl reference. Values are reported vs Ag/AgCl.

Figure 4. Cyclic voltammogram of (A) K2[Fe(DMB)(CN)4] and (B)K2[Fe(BMB)(CN)4] adsorbed to a mercaptoundecanoic acid SAM ona gold ball electrode. The cyclic voltammograms were collected at 100

mV/s in 100 mM PBS at pH 7.2 with Ag/AgCl as a reference.




8) is mainly positive in nature. This portion of the protein shouldhave favorable interactions with the net negative charge of 1and 2.

DISCUSSION

The redox properties of metal complexes in proteins areinfluenced by changes in the surrounding protein environment(e.g., cytochrome c and blue copper proteins) (48-50). Metalcofactors bound to proteins experience a solvation environmentwith a lower local dielectric constant than that of aqueous buffer.The dielectric constant (ε) of water is 80, whereas ε is estimatedto be between 4 and 20 inside a protein (51-53). Changesin the dielectric constant affect the energetics for electronictransitions of metal redox centers. Further, the protein impartsa structured charge environment around the metal complex dueto the partial charges of each atom. This charge structure, withits partial positive or negative properties, stabilizes more stronglyeither the reduced or oxidized state of the metal. Finally, theprotein can be viewed as a scaffold containing electrophilic ornucleophilic residues capable of multiple interactions with themetal cofactor.

In order to probe changes in outer-sphere coordinationinduced by changes in ligand-receptor binding, we havesynthesized a biotinylated iron(II) complex (1) and a desthio-biotinylated iron(II) complex (2). These redox-active, solvato-

chromic transition metal complexes are designed to bind to theprotein avidin. Biotin and desthiobiotin bind to avidin with K a’sof 1015 M-1 and 1013 M-1, respectively (54). Therefore, weakerligand-receptor binding is expected to give the [Fe(DMB)-(CN)4]2- complex slightly more flexibility within the bindingpocket as compared to [Fe(BMB)(CN)4]2-. Therefore, thedielectric constant, charge structure, van der Waals, and

Figure 5. Absoprtion spectroscopy of K2[Fe(BMB)(CN)4] and K2[Fe(DMB)(CN)4] in increasing concentrations of DMF in PBS buffer at pH 7.2.(A) Representative UV-vis spectra of K2[Fe(DMB)(CN)4]. (B) Correlation of the K2[Fe(BMB)(CN)4] MLCT bands. (C) Correlation of theK2[Fe(DMB)(CN)4] MLCT bands with increasing amounts of DMF. The wavelengths of the MLCT bands are linearly correlated to the solventfraction indicating that no preferential solvation of the complexes occurs.

Table 4. Binding Constants and Enthalpy of Binding for Iron(II)Complexes

ligand K (M-1) ∆ H (kcal/mol)

K2[Fe(BMB)(CN)4] 3.5 ×107-104

K2[Fe(DMB)(CN)4] 1.5 ×106-80

Table 5. Data Collection, Phasing, and Refinement Statistics

data collection

beamline DND-CAT (Sector 5)wavelength (Å) 1.279resolution range (Å)a 30-3.1 (3.27-3.10)total observations 64 647 (9347)unique observations 9587 (742)completeness (%) 99.9 (100)redundancy 12.3 (12.6)

I / σ 28.0 (5.7) Rsym

b (%) 9.9 (40.6)

Refinementno. of reflections, working/test 8292/975

Rwork (%)c 25.5 Rfree (%)d 33.0Protein atoms 1856ligand atoms 61

water molecules 22r.m.s.d. bond length (Å) 0.009r.m.s.d. bond angle (°) 1.4average B-value (Å2) 65.5

a Values in parentheses are for the highest-resolution shell. b Rsym )

Σi Σhkl | I i(hkl) - ⟨ I (hkl)⟩|/ Σhkl ⟨ I (hkl)⟩, where I i(hkl) is the ith measureddiffraction intensity and ⟨ I (hkl)⟩ is the mean of the intensity for theMiller index (hkl). c Rwork ) Σhkl |F o(hkl)| - |F c(hkl)|/ Σhkl|F o(hkl)|. d Rfree

) Rwork for a test set of reflections (5%).




hydrogen bonding contacts near the metal will differ for 1 and2. We have used a variety of techniques to observe differencesin the electronic structure of these complexes as imparted bythe protein.

Electronic Structure, Solvatochromism, and AcceptorNumber. Iron(II) tetracyano complexes are known to be highlysensitive to changes in environment (4, 27, 29, 55, 56 ).[Fe(bpy)CN)4]2- is a paramagnetic complex with C 2V symmetrythat has completely filled dπ orbitals (d xy, d xz, d yz), and theseorbitals act as the HOMO set for the molecule. Oxidation of

the iron(II) to iron(III) species requires removal of an electron

from one of these metal-centered orbitals.The environmental sensitivity of these complexes is largely

due to noncovalent interactions with the lone pair electrons onthe cyano ligands. Solvent molecules interact noncovalently withthese lone pairs. Strong electron acceptors such as waterdecrease the electron density at the CN and therefore increaseπ backbonding with the metal ion. This interaction stabilizesthe iron(II) state with respect to the iron(III) state. Dependingon solvent, the LUMO in [Fe(bpy)(CN)4]2- might be either theunfilled metal d-orbitals (d z2 and d x2- y2) or the π * levels of thebipyridine ligands (28). The first optically allowed transitionsfrom the dπ orbitals, however, are to the π * orbitals of thebipyridine ligand (47 ). Weak acceptors such as DMF decreasethe π backbonding as compared to water. Therefore, as DMFis added to the system, water molecules are displaced and theiron(II) state becomes destabilized. A solvent with a highacceptor number such as water will result in a higher-energymetal to ligand charge transfer (MLCT) as compared to weakacceptors such as DMF. These changes in MLCT energy aresignificant enough to be observed spectroscopically. Likewise,stabilization of the iron(II) complex will result in higheroxidation potentials that can be observed by electrochemicalmethods such as cyclic voltammetry.

The absorbance of 1 in various solvents was measured toinvestigate the sensitivity of the physical properties of 1 tosecond-sphere effects. The solvents were chosen to represent arange of ANs. We observed a larger shift of the low-energyMLCT1 band compared to the MLCT2 band. This resultindicates that the solvent is interacting with both the cyano andbipyridine ligands to stabilize the π * orbitals of the bipyridine

Figure 6. Structure of [Fe(BMB)(CN)4]2- bound to avidin. (A) The asymmetric unit shows [Fe(BMB)(CN)4]2- bound to two avidin protomers. Theother two protomers are related by crystallographic symmetry. 2|F o| - |F c| omit maps are depicted around the modeled ligand at 1σ (green) and 4σ (red). (B) View of ligand-protein interactions. Amino acids in green interact with biotin as observed in crystal structures of native D-biotin boundto avidin (26 ). Residues in gray hydrogen bond to the [Fe(bpy)(CN)4] portion of the complex. The 2|F o| - |F c| omit density is superposed at 4σ .

Figure 7. A view of the final structure of the molecular dynamics simulation of avidin bound by (A) [Fe(BMB)(CN)4]2- and (B) [Fe(DMB)(CN)4]2-.One monomer of the tetramer avidin is shown.

Figure 8. Isopotential surfaces of the avidin binding pocket (shown inspace filling mode) as calculated by the program HARLEM. Bluecorresponds to positive charges, and red corresponds to negativecharges. This representation indicates that there are favorable interac-tions between the binding pocket and the negatively charged 1 (shown)and 2.




ligand to varying degrees. This phenomenon has been describedfor cyanoiron(II) complexes by Toma and co-workers anddemonstrates that changes in the second-sphere environmentaround any part of the molecule, whether it is the bipyridine orthe cyano ligands, result in spectroscopic changes (56 ).

As increasing amounts of DMF are added to an aqueoussolution of 1 or 2, the energy of MLCT bands decreases linearly(Table 2, Figures 3 and 5). This linear correlation in a binarysolvent system demonstrates the effect of an increasingly

nonpolar environment and indicates that no preferential solvationof the complexes occurs (56 ). DMF displaces water moleculesfrom the second sphere of the metal complex, and the complexesdo not have a tendency to maintain a solvation shell of waterwhen a nonpolar solvent is introduced.

To investigate the sensitivity of the redox potentials of 1 and2 to second-sphere coordination, the electrochemical potential

E 1/2 of 1 and 2 was determined in a variety of solvents. As theAN is decreased, the E 1/2 decreases (Table 1, Figure 2). Further,as increasing amounts of DMF are added to an aqueous solutionof 1 or 2, the E 1/2 decreases linearly (Table 2, Figures 3 and 5).

These solvent experiments provide a basis for rough com-parison with avidin-binding results. Upon addition to an aqueoussolution of 1 or 2, DMF replaces water in the vicinity of the

metal complex; when 1 or 2 binds to avidin, water will bedisplaced from the metal center in a similar way.X-ray Crystal Structure and Binding Properties. Isother-

mal titration calorimetry was used to measure the bindingconstants of 1 and 2 to avidin. The binding affinities of 1 and2 for avidin are 3.5 × 107 M-1 and 1.5 × 106 M-1, respectively.These values demonstrate a significant decrease in bindingaffinity compared to that of native D-biotin (K ) 1 × 1015 M-1)and D-desthiobiotin (K ) 1 × 1013 M-1). The data fit best tothe case where all four binding sites are identical, and bindingis independent. Previous studies by Livnah and co-workersdemonstrate that when bound to avidin, modification of biotinat the carboxyl terminus by a caproic acid results in disruptionof the hydrogen bonding of Thr 38, Ala 39, and Thr 40 with

the carboxyl terminus of biotin. This disruption results in adecrease in binding affinity (7 to 8 orders of magnitude) forbiotin analogues that have an amide at the carboxylic acid (57 ).

The crystal structure shows two unique protomers (Figure6). Avidin is a tetramer; the other two protomers are related bycrystallographic symmetry. It is apparent that 1 is in a relativelyhydrophobic (low dielectric) environment and interacts directlywith the protein via electrostatic and hydrogen bonding interac-tions. While several biotinylated-transition metal complexeshave been synthesized, only one other has been structurallycharacterized bound to avidin (20, 23, 58-61). Similar to theobservations of Ward et al., there are short contacts betweenthe metal center of 1 and amino acid residues. Importantly, thereis no major reorganization of the host protein.

The three-dimensional structure of 1 bound to avidin showsthat the cyano ligands of the iron complex interact with specificresidues in the avidin binding pocket. In one protomer, the Ser41 residue of the L3,4 loop interacts directly with a CN groupon the iron(II) complex, and Ser 102 of the L5,6 loop interactswith a CN group through a water molecule. We expect theseinteractions with the cyano groups to have an observable effecton the electrochemical properties of 1 and 2 bound to avidin.The stabilizing, attractive interaction of the protein with thecyano groups prevents the loops from opening as they do inthe cases where the carboxyl terminus is derivatized by caproicacid (40, 57, 62).

In the second protomer, electron densities for the [Fe-(bpy)CN)4]2- moiety of 1 and residues 41-44 on the L3,4 loopwere not observed. These differences are most likely due todisorder imposed by crystal packing. This conclusion is sup-

ported by ITC data that are best fit for a system with fouridentical binding events.

We have used the program HARLEM to calculate theisopotential surface of avidin and show that the binding pocketof avidin is positive in nature (Figure 8). 1 and 2 are negativelycharged and have small polar ligands that should promoteinteractions with the protein. This portion of the protein shouldhave favorable interactions with the net negative charges of 1and 2.

Molecular Dynamics Simulations. Molecular dynamicssimulations were carried out with 1 and 2 bound to avidin tocompare the local environments of the transition metal com-plexes when bound to avidin. Several similar conformationswere found to be accessible to 1 and 2 when bound to avidin inthe course of the molecular dynamics simulation (conformationsof 1 shown in Supporting Information Figure S2). The confor-mation in closest agreement with the crystal structure of 1 boundto avidin was chosen to compare the second-sphere environmentof 1 and 2 when bound to avidin. For comparison, the distancebetween the apex of loop 3,4 (Ser 41) and the metal center wasmeasured. This distance is specifically of interest because thecrystal structure shows that Ser 41 has a direct interaction with1. The distance from the R-carbon of Ser 41 to the iron center

in 1 is 5.76 Å, whereas in 2, the distance is 5.92 Å. We did notobserve a water molecule between Ser 102 and the cyano groupin the molecular dynamics simulation, as was observed in thecrystal structure of avidin with 1. The MD simulations showthat Ser 101 has a stronger interaction with the binding ligandthan does Ser 102. The distance between Ser 101 and the ironcenter is ∼5 Å, while the distance between Ser 102 and theiron atom is ∼10 Å. These observations are slightly differentthan values determined from the crystal structure; however, theyare reasonable due to the fluxional nature of the protein in themolecular dynamics simulations. While the location of the ligandwithin the protein in the crystal structure is very informative, itis only a snapshot of the ensemble of configurations that themetal complex samples during a spectral or electrochemical

measurement. The MD simulations further show that, when 2is bound, the protein structure is more disordered. Figure 7shows the disorder in strands 3 and 4 and loops 7,8 and 5,6 for2 as compared to 1 bound to avidin. The environment of 2 isslightly more open and water-accessible than the environmentthat 1 experiences.

Electronic Affects of Avidin Binding. UV-vis spectroscopywas used to measure roughly the HOMO-LUMO gaps of 1and 2 while bound to the protein. The optical spectra in aqueoussolution show two MLCT bands, one to each of the first twoπ * levels of the bipyridine ligands (MLCT1 at lower energies,480 nm for 1 and 486 nm for 2; and MLCT2 at higher energies,341 nm for 1 and 345 nm for 2). Upon binding of avidin to 1,the MLCT1 band red-shifts 20 nm and the MLCT2 band red-

shifts 7 nm. Upon binding of avidin to 2, the MLCT1 bandred-shifts 10 nm and the MLCT2 band red-shifts 6 nm. Whenan excess of biotin is added to displace 1 or 2 from avidin, the λmax of the MLCT bands shifts back to the original valuesreported for the compounds in aqueous solution. These resultsdemonstrate that the outer-sphere coordination of the moleculechanges with the ligand receptor binding. The UV-vis studiesshow that the molecule with the lower binding constant (2) hassmaller changes in MLCT bands compared to that of 1,consistent with a higher acceptor number environment.

The spectroscopic shifts upon avidin binding to 1 and 2 aresimilar to the shifts observed when increasing amounts of DMFare added to aqueous solutions of 1 or 2. The protein bindingevent displaces water molecules from the metal center just asDMF replaces water molecules that are interacting with themetal center. This solvent system provides a convenient model




for systematically varying the environment to crudely mimicprotein binding.

Electrochemistry. The redox potentials of 1 and 2 whilebound to avidin were found to shift by -140 and -127 mV,respectively. These potentials were measured by electrostaticallyadsorbing the protein to a monolayer of mercaptoundecanoicacid (MUA). These dramatic shifts are most likely due to theinteraction of the complex with the monolayer as well as theprotein. Being sandwiched between the protein and the mono-layer minimizes any interaction with bulk water and places thecomplexes in an even lower dielectric environment comparedto avidin in solution. The protein/monolayer combinationdestabilizes the iron(II) state with respect to the iron(III) state,compared with the complex in aqueous solution, raising theenergy level of the orbitals that comprise the HOMO. TheHOMO is destabilized by 1130 cm-1 and 1050 cm-1 for 1 and2, respectively.

Immobilization techniques are commonly used to studyelectroactive proteins (61, 63-70). In our case, a monolayer of MUA was formed and then washed with NH4OH(aq) in order toleave a number of negatively charged acids on the surface (thepI of avidin is ∼10, and therefore the protein should bepositively charged at pH 7). Figure 8 shows the chargedistribution at the binding pocket of avidin at pH 7. There is asignificant area with a positive charge near the binding site.This positive charge ensures that a portion of the avidin on themonolayer will be oriented so that the bound iron complexeswill make effective contact. The observed electrochemical signalis likely from these bound complexes (61). Since avidin is atetramer, it is likely that a maximum of two of the binding sitesare in contact with the monolayer at any given time.

CONCLUSION

We have established that the electronic structures of [Fe-(BMB)(CN)4]2- and [Fe(DMB)(CN)4]2- are highly sensitive tononcovalent interactions. The optical spectrum shifts up to 20nm in organic solvents with lower acceptor numbers than water.

The shift is linearly dependent on the solvent acceptor numberindicating there are specific interactions between the solventand the CN and bpy ligands. Similarly, the electrochemicalresponse of 1 and 2 in various organic solvents shows a shiftthat is linearly dependent on solvent AN; shifts of up to 150mV are observed. These data suggest that the electronic structureof 1 and 2 is sensitive to second-sphere coordination changesand solvation.

The results from solvent studies confirm that the complexescan be used as sensitive probes to investigate the electronicproperties of protein binding sites. Binding of 1 and 2 to avidinresults in visible changes in the electronic spectra. The shiftsare similar to those observed in the solvent studies. We expectthe binding event to exclude water from the metal complex,resulting in a lower AN environment. For example, binding of 1 to avidin can be compared to a solution of 1 in 23% DMF inwater.

Our data show that the environments of 1 and 2 in avidinare observably different. ITC measurements show that thebinding of 2 is weaker than that of 1 by a factor of ∼20.Therefore, we expect 1 and 2 to sample slightly differentenvironments in the binding pocket. Indeed, small differencesare observed in both the optical and electrochemical data. Uponbinding to avidin, the optical spectrum shifts 20 and 7 nm for1, while the same transitions shift 10 and 6 nm for 2. Thebinding of 2 can be compared to 12% DMF in water. Thedifference in redox potential shifts is small (10 mV), yet wellabove the error for such a measurement.

The three-dimensional X-ray structure of 1 bound to avidinshows 1 partially buried in a hydrophobic pocket with a direct

interaction between the protein and the transition metal complex.An open-loop conformation, expected from the presence of theamide, would prevent the avidin from shielding the complexfrom bulk water and minimize the observable change inelectrochemical potential and absorbance properties. Isopotentialcalculations, however, show a patch of positive charge near thebinding site that should favor binding of the negatively charged1 and 2. These interactions may partially counteract the effectsof having the amide in place of the carboxylic acid. Finally,molecular dynamics simulations show that, when 2 is bound toavidin, the protein structure is slightly more disordered.However, for the MD snapshot most resembling the crystalstructure, the distances between the metal center and selectresidues are nearly the same as when 1 is bound.

Together, these results demonstrate that even small changesin the binding of a ligand (1 or 2) result in small yetexperimentally observable changes in second-sphere coordina-tion imposed by the protein. Specifically, these changes includethe partial charge structure, van der Waals interactions, andhydrogen bonds. Small changes in these weak interactions arepowerful enough to induce changes in the electrochemical andoptical properties of the metal cofactors.

The synthetic versatility and the sensitivity of [Fe(bpy)-(CN)4]2- complexes to local environmental changes makes thesecomplexes candidates for use in electronic and optical proteindetection devices for any number of ligand-receptor pairs. Forexample, by systematic change of the protein binding ligand(e.g., adding or removing hydrogen bond donors) and observingthe effect of protein binding on the spectroscopic properties,the nature of an unknown binding pocket can be evaluated.Further, the shift in the electrochemical potential indicateswhether solvent is being excluded from the metal center andtherefore can be used to evaluate the hydrophobicity of thebinding site.

Sensitivity to small changes in binding affinities indicates thatthese species can be used as probes for remotely evaluating thenoncovalent factors that affect ligand-receptor binding. Ourfuture studies will translate this approach to self-assembledmonolayer systems for investigating the effects of proteinbinding on the electrochemical properties of the metal complex.

ACKNOWLEDGMENT

We thank Belovo, Inc. for providing deglycosylated avidin.We thank Harry Gray, Igor Kurnikov, Baudilio Tejerina, andAlec Durrell for helpful discussions. This work was funded bythe Nanoscale Science and Engineering Initiative of the NationalScience Foundation under NSF Award Number EEC-0647560and Ohmx Corporation. MR thanks the MURI program of theDoD for support.

Supporting Information Available: Molecular modeling

overlay with crystal structure, isothermal titration calorimetrydata, cyclic voltammograms of 1 in 0-50% DMF/water. Thismaterial is available free of charge via the Internet at http:// pubs.acs.org.

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