Oct 15, 2018 - of the two reagents were chosen to ensure that any net electron transfer reactions to or from the limiting dicy- anobis( 1,lO-phenanthro1ine)iron ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 266, No. 29, Issue of October 15, pp. 19203-19211, 1991 Printed in U.S.A.
0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Mixed Ligand Complexesof Iron with Cyanide andPhenanthroline as New Probes of Metalloprotein Electron Transfer Reactivity ANALYSIS OF REACTIONS INVOLVING RUSTICYANINFROM THIOBACILLUS FERROOXIDANS* (Received for publication, April 2, 1991)
Robert Blake 11, Kathy J. White, and Elizabeth A. Shute From the Department of Biochemistry, Meharry Medical College, Nashville, Tennessee 37208
A family of 12 different mixed ligand complexes of iron with cyanide and substituted 1,lO-phenanthroline was prepared. The electron transfer properties of each reagent were systematically manipulated by varying the substituent(s) on the aromatic ring system and the stoichiometry of the two types of ligands in the complex. Values for the standard reduction potentials of each member of this family of electron transfer reagents weredetermined and spanned from 500 to 900 mV. The one-electron transfer reactions between each of these substitution-inert reagents and the high potential blue copperprotein, rusticyanin, from Thiobacillus ferrooxidans were studied by stopped flow spectrophotometry under acidic conditions. For comparison with the protein results, the kinetics of electron transfer between each of these reagents and sulfatoiron were also investigated. The Marcus theory of electron transfer was successfully applied to this set of kinetic data to demonstrate that 10 of the 12 reagents had equal kinetic access to the redox center of the rusticyanin and utilized the same reaction pathway for electron transfer. The utility of these synthetic electron transfer reagents in characterizing the electron transfer properties of very high potential, redox-active metalloproteins is illustrated.
isolated from Thiobacillus ferrooxidans, an acidophilic chemolithotroph that grows autotrophically on ferrous ions (1619). The protein is thought to be expressed into the periplasmic space of this Gram-negative bacterium and to play an important, although as yetundefined, role in the iron respiratoryelectron transport chain. The rusticyanin possesses two functional characteristics that distinguish it from other comparably sized type I copper proteins: it is redoxactive only in acidic solutions, down topH 0.2; andits reduction potential is 680 mV (20), avalue much higher than that of the average value of 210 mV (21) attributed to other members of this arbitrary class of copper metalloproteins. Previous kinetic studieson theelectron transfer properties of purified rusticyanin utilized sulfatoiron, an inorganic complex of physiological significance to the intact organism (22-24). Aprincipalbarrier tofurther probes of the rusticyanin’s electron transfer reactivity is that relatively few well characterized inorganic or small organometallic complexes are availablewith theappropriate electrochemical, solubility, and structural properties that arerequired to probe a redox center of such high reduction potential under strongly acidic conditions. The present paper describes the synthesis, characterization, and electron transfer properties of a series of mixed ligand complexes of Fe(I1) and Fe(II1) with cyanide and 1,lO-phenanthroline. By introducingdifferent substituents on the aromatic diimine and varying the stoichiometry of the substiExtensive kineticdata areavailable on theelectron transfer tuted phenanthroline in the final organoiron complex, a series reactions of selected inorganic and small organometallic comof substitution-inert electron transfer reagents were conplexes with isolated electron transport proteins,especially the structed with reduction potentials from 500 to 900 mV. Kicytochromes (1-4), type I blue copper proteins (5-8), and netic and thermodynamicstudies on the electron transfer various iron sulfurproteins (9-12). One goal of such research reactions of each of these substitution-inert iron complexes has been the elucidation of reaction pathwaysavailable to the withbothsulfatoiron and rusticyanin are presented. The proteins and the manner in which the polypeptide envelopes family of electron transfer reagents described herein could have modified the inherent reactivity of the metal center. In prove useful in probing the electron transfer reactivity of this particular, the Marcus theory of electron transfer has been novel class of high potential respiratory proteins. widely employed to deduce, from the relevantkinetic and thermodynamic data, thefactors governing the ratesof outer EXPERIMENTALPROCEDURES AND RESULTS’ sphere electron transfer reactions between small organometallic complexes and metalloproteins (13-15, and references Absorbance Properties-Both the redox potential and the therein). A wide assortment of well characterized outer sphere electron transfer reactivity of the ferrous/ferric couple are electron transfer reagents varyingin charge, reductionpoten- profoundly influenced by the ligands coordinated to the iron tial, andligand structure areavailable to systematically probe cation. Hexoaquoiron has a reduction potential of 770 mV the reactive sites of metalloproteins that have reduction po- and reacts relatively slowly in its electron transfer reactions tentials in theapproximate range of 0-400 mV. both with itself (self-exchange electron transfer) and with Rusticyanin is a 16.3-kilodalton, type I blue copper protein other electron transfer reagents. The conjugate base of hydro*This research was supported by Grant DE-FG05-85ER13339 from the United States Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
’
Portions of this paper (including “Experimental Procedures,” part of “Results,” Figs. 2 and 4-6, Tables 1-111, and Equations 4-8) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Fullsize photocopies are included in the microfilm edition of the Journal that isavailable from Waverly Press.
19203
Electron Transfer between Rusticyanin and Cyano(phenanthro1ine)iron
19204
gen cyanide binds much more strongly to ferric ions than it does to ferrous ions. This discriminatory binding serves to make the ferrous ion more reducing; hexacyanoferrate thus has a muchlower reduction potential, 410mV, than does hexoaquoiron. Conversely, the aromatic diimine 1,lO-phenanthroline binds much morestrongly to theferrous ion than it does to ferric ions, with the consequence that tris(1,lOphenanthro1ine)iron has a much higher reduction potential, 1,060 mV, than does hexoaquoiron. As part of an effort to devise substitution-inert electron transfer reagents with reduction potentials intermediate between those of 410 and 1,060 mV, a series of mixed ligand complexes of iron with cyanide and 1,lO-phenanthroline were constructed by varying the stoichiometry of the two types of ligands in the final six-coordinate organoiron complex. The visible absorbance spectra of a family of such complexes is shown inFig. 1. As illustrated in the figure, the complex between Fe(I1) and 1,lO-phenanthroline exhibits a red color whose intensity depends on the number of phenanthrolines cyano(1,lOin the complex. Curves a, b, andcrepresent phenanthroline)iron(II) complexes containing 3, 2, and 1 equivalents of 1,lO-phenanthroline, respectively. Hexacyanoferrate(II), containing no phenanthroline ligand, has negligible absorbance over the same range of wavelengths (data not shown). A series of five additional families of cyano(lJ0phenanthro1ine)iron complexes wereobtained using 1,lO-phenanthroline derivatives that contained electron donating or withdrawing substituents on the aromatic rings. The wavelengths of maximum absorbance in the visible region, along with the corresponding molar absorption coefficients, of the ferrous forms of each of the 12 cyano(1,lO-phenanthro1ine)iron complexes synthesized here are summarized in the first two columns of Table I (Miniprint). The visible absorbance spectrum of each compound was substantially bleached upon the one-electron oxidation of the ferrous complex. The absorbance difference between the reduced and the oxidized forms of each cyano(1,lO-phenanthro1ine)ironcomplex thus constitutes an intrinsic spectrophotometric probe whereby the transient changes in the redox state of the population of molecules can be monitored with great sensitivity. Electron Transfer with Sulfatoiron-The object of these experiments was to obtain detailed kinetic data on the oneelectron transfer reactions between cyano(lJ0-phenanthro-
-I E -
12.5
0
‘310.0 E t
c
.-0 ) 7.5 0
.-
-4-
& Q)
8
5.0
c
0 .c
2 2.0
w
4
a
0
400
500
600
1ine)iron and soluble iron in acidic solutions in the presence of an excess of sulfate. Under these experimental conditions, the principal form of the iron in solution is the soluble sulfatoiron complex (29). When each of the 12 cyano(1,lOphenanthro1ine)iron derivatives in Table I was mixed in a stopped flow spectrophotometer with a 4-fold or greater molar excess of soluble sulfatoiron (pseudo-first order conditions), each kinetic trace of the change in absorbance couldbe described mathematically as a single exponential function of time. Accurate values of both the amplitude and the pseudofirst order rate constant for each absorbance change were obtained from each kinetic trace as described previously (22). As long as pseudo-first order conditions were maintained, a 10-fold variation in the concentration of the particular cyano(1,lO-phenanthro1ine)ironcomplex affected only the amplitudes of the observed spectral changes, not the values of the corresponding pseudo-first order rate constants. The amplitudes were directly proportional to theconcentration of the cyano(1,lO-phenanthro1ine)iron. Representative examples of the dependence of the pseudofirst order rate constants for the oxidation of selected dicyanobis(1,lO-phenanthroline)iron(II)complexes upon the total concentration of Fe(II1) are presented in Fig. 2A (Miniprint). A typical kinetic study for the oxidation of these organoiron(I1) derivatives employed at least five different concentrations of soluble Fe(II1) that evenly spanned a 16fold range in concentrations. The dependence of the pseudofirst order rate constantsfor the reduction of the corresponding dicyanobis(1,lO-phenanthroline)iron(III)complexes upon the total concentration of Fe(I1) is presented in Fig. 2B. A typical kinetic study for the reduction of these organoiron(II1j derivatives employed at least four different concentrations of soluble Fe(I1) that spanned a &fold range in concentrations. Regardless of the direction of electron flow, values of the pseudo-first order rate constants for each electron transfer reaction were directly proportional to the concentration of excess soluble iron in all cases; no convincing evidence for rate saturation was obtained in any of these studies. The value of the slope of each line in Fig. 2 represents an apparent second order rate constant for the electron transfer reaction between the organoiron derivative and thesoluble iron. Values of these second order rate constants were obtained for both the Fe(II1)-dependent oxidation andthe Fe(I1)-dependent reduction of each of the 12 cyano(1,lO-phenanthro1ine)iron compounds investigated andare summarized in Table I1 (Miniprint). The kinetic data summarized in Table I1 were exploited to estimate the reduction potential of each cyano(1,lO-phenanthro1ine)iron complex. Each substitution-inertorganoiron complex featured in Table I1 would be expected to transfer an electron by an outer spherebimolecular reaction. An outer sphere electron transfer reaction is one in which the two reactants do not share a common atom or group, or more generally, one in which the interaction of the relevant electronic orbitals of the two centers is weak. Extensive experimental data on a variety of known outer sphere electron transfer reactions have established that thekinetic and thermodynamic properties of the reaction partners may frequently be correlated by the “cross-reaction” equation developed by Marcus (13-15):
Wavelength, nm kxv = Jkxx k w KXY (1) FIG.1. Absorbance spectra of tris(1,lO-phenanthro- where kxy is the second order rate constant for the transfer line)iron(II) (a),dicyanobis(1,lO-phenanthroline)iron(II)( b ) , and tetracyano(1,lO-phenanthroline)ferrate(II) (c). Spectra of an electron from X to Y, kxx and k y y are the two selfwere recorded a t 25 “C in 0.01 N sulfuric acid,pH 2.0. Each absorption exchange rate constants (e.g. for the transfer of an electron from one molecule of X to another molecule of X), and KXY coefficient is the average of four determinations.
Electron Transfer between Rusticyanin and Cyano(phenanthro1ine)iron is the equilibrium constant for the electron transfer. Each self-exchange rateconstantisa measure of the intrinsic reactivity of each reactant and isrelated to theenergy barrier created by the internal and solvent nuclear rearrangements that must occur immediately prior to actual electron transfer. Equation 1 is a much simplified version of the theoretical treatment developed by Marcus for outersphere electron transfer reactions, butit has nonetheless been shown to correlate a large body of kinetic and thermodynamic data, particularly in those electron transfer reactions of an acceptor with a series of related donors (or vice versa). For the reverse of the reaction represented by Equation 1, the transfer of an electron from Y to X, it follows that kux = Jkxx kuu Kux
0.4
$ 0.3 C 0
e
5:
D
0.2
4
0.1
(2)
where kYX is the second order rate constant for the transfer of an electron from Y to X, Kyx is the corresponding equilibrium constant, and kxx and kyy are defined above. Recognizing thatKXy= l/Kyx, Equation1may be divided by Equation 2 and the dividend rearranged to yield kxu Kxy = kux
19205
(3)
It is satisfactory, and indeed necessary, that the Marcus expressions for the forward and backward electron transfer reactions should satisfy the thermodynamics of the system! The kinetic data in Table IT were used in accordance with Equation 3 to calculate an equilibrium constant for the electron transfer between sulfatoiron and each cyano(1,lO-phenanthro1ine)iron complex investigated. Sulfate binds preferentially to the ferric form of soluble iron, thereby effectively lowering the value of the reduction potential for the ferric/ ferrous couple. Under the solution conditions imposed on the kinetic experiments summarized in Table 11, the effective reduction potential of the sulfatoiron in each case was calculated tobe 710 mV(22). Using this potentialof the sulfatoiron as a point of reference, the reduction potential of each cyano(1,lO-phenanthro1ine)ironcompound in Table I1 was then determined. The value of each reduction potential determined from the kinetic studies described abovewas then verified by independent equilibrium experiments that employed soluble iron and the cyano(1,lO-phenanthro1ine)ironcomplex of interest. A representative example of such an equilibrium experiment is illustrated in Fig. 3. Each absorbance spectrum in Fig. 3 was takenaftera small concentration of dicyanobis(1,lOphenanthroline)iron(II) was introduced into a solution that contained an excess of soluble sulfatoiron. The concentrations of the two reagents were chosen to ensure thatany net electron transfer reactions to or from the limiting dicyanobis( 1,lO-phenanthro1ine)ironwould have a negligible effect on the relative concentrations of the ferrous and ferric forms of the excess sulfatoiron. Each spectrum in Fig. 3 provided a sensitive means of quantifying the fractions of the total population of dicyanobis(1,lO-phenanthro1ine)ironmolecules that were either oxidized or reduced. By systematically varying the ratio of sulfatoiron(II)/sulfatoiron(III), the redox state of the population of dicyanobis(1,lO-phenanthro1ine)iron molecules was systematically manipulated to yield the standardNernst plot shown in the inset in Fig. 3. A value of 800 mV for the reduction potential of dicyanobis(1,lOphenanthro1ine)iron was obtained from the abscissa intercept of the Nernst plot. Similar equilibrium experiments were performed on each of the 12 cyanobis(1,lO-phenanthro1ine)iron compounds investigated. Acceptable agreement (within 5-15 mV) was observed between the value of each
0
400
500
600
Wavelength, nm FIG. 3. Absorbance spectra of dicyanobis(1,lO-phenanthro1ine)iron in different mixtures of eulfatoiron(I1) and sulfatoiron(II1). Final concentrations: dicyanobis(1,lO-phenanthroline)iron, 100 p M ; total soluble iron, 20 mM; and sulfate, 0.1 M. The ratios of sulfatoiron(II)/sulfatoiron(III) were 0.0, 6.8, 14.6, 23, 40, 65, 120, and m in experiments a-h, respectively. Inset, a standard Nernst plot of the reductive titration of dicyanobis(1,lO-phenanthro1ine)iron. f”’ and Y dare the fractions of the dicyanobis(1,lO-phenanthro1ine)iron in the oxidized and reduced states, respectively. E,, is the total system redox potential. The ordinate intercept and slope were determined by a linear regression analysis.
reduction potential obtained by this equilibrium method compared with that obtained by the kinetic method described above. The mean values of the reduction potentials obtained by the two methods are listed in the last column of Table I. Although very little data on the redox properties of these compounds are available for comparison from the literature, the reduction potential of tetracyano(lJ0-phenanthro1ine)iron has been reported by two independent laboratories to be 570 (30) and 560 mV (31), respectively, in agreement with the value determined here. Electron Transfer with Rusticyanin-The object of these experiments was to obtain detailed kinetic data on the oneelectron transfer reactions between cyano(1,lO-phenanthro1ine)iron and purified rusticyanin in the acidic sulfate solutions usually employed for functional studies of the purified protein. The visible absorbance spectrum of oxidized rusticyanin exhibits a prominent peak at around 600 nm that disappears upon the one-electron reduction of the protein (22). The redox-dependent absorbance properties of the rusticyanin thus permit transient changes in the redox state of the protein to be monitored with satisfactory sensitivity. When oxidized or reduced rusticyanin was mixed ina stopped flow spectrophotometer with a molar excess of a cyano(1,lOphenanthroline)iron(II) or corresponding cyano(1,lO-phenanthroline)iron(III) compound, respectively, each kinetic trace of the change in absorbance at 597 nm could be described mathematically as asingle exponential function of time. Representative kinetic traces of the tetracyano(5-nitro-1,lO-phenanthrolinelferrate(I1)-dependent reduction of rusticyanin are shown in Fig. 4 (Miniprint). These datarepresent the fastest electron transfer reactions that were monitored in direct mixing experiments for the current study. Examples of the dependence of the pseudo-first order rate constants for the reduction and oxidation of rusticyanin upon the concentration of selected cyano( 1,lO-phenanthroline) complexes of iron are presented in Fig. 5, A and B, respectively (Miniprint). Values of the pseudo-first order rateconstants for each electron
19206
Electron Transfer between Rusticyanin
transfer reaction were directly proportional to theconcentration of excess organoiron reagent in all circumstances. Values for the second order rate constants for each electron transfer reaction were obtained from the slopes of linear plots such as those illustrated in Fig. 5 and are summarized in Table I11 (Miniprint). Many of the bimolecular electron transfer reactions between rusticyanin and individual cyano(1,lO-phenanthro1ine)iron complexes were too rapid to be accurately investigated by direct mixing experiments in the stopped flow spectrophotometer. Noting that thebimolecular electron transfer reactions of cyano(1,lO-phenanthro1ine)iron with either soluble iron (Fig. 2) or rusticyanin (Fig. 5) were reasonably rapid, limiting concentrations of selected cyano(1,lO-phenanthro1ine)iron complexes were exploited to catalyze the relatively sluggish electron transfer reactions between soluble iron and purified rusticyanin. The examples in Fig. 6 (Miniprint)illustrate theapproach adopted to estimate the second order rate constants for these extremely rapid electron transfer reactions. Acceptable agreement, within &lo%,was obtained between those values of electron transfer rate constants determined by direct stopped flow spectrophotometric measurementsandthe corresponding values determined by the indirect steady-state kinetic approach outlined in the Miniprint. The lattersteady-state approachwas therefore applied to each of the bimolecular electron transfer reactions between rusticyanin and individual cyano( 1,lO-phenanthro1ine)iron complexes that was too rapid to be investigated by direct mixing experiments in the stopped flow spectrophotometer. The values of the second order rate constantsobtained by the steady-state approach are included in Table 111. The kinetic data summarized in TableI11 were exploited to estimate the reduction potential of the rusticyanin. It is evident from Equation 3 that
and Cyano(phenanthro1ine)iron
I
-200 I
500
600
I
I
I
I
700
800
900
ReductionPotential, mV FIG. 7. Kinetic determination of the reduction potential of rusticyanin in 0.1 M sulfate at pH 2.0. Each datum represents kinetic and thermodynamic data collected using either a tetracyano(lJ0-phenanthro1ine)iron (open circles) ora dicyanobis(1,lOphenanthro1ine)iron (closed circles) derivative. Values of k,,* and kwd were taken from Table 111. Corresponding values for the reduction potential of each compound were taken from Table I. The slope and abscissa intercept were determined by a linear regression analysis.
transfer between the rusticyanin and the soluble iron and thus permit rapid equilibration of the available electrons among the individual redox partners. The concentrations of the three reaction partners were chosen to ensure that any -kox = [CPI(II)][RCu(II)] KRCu(I)CPI(III) = net electron transfer reactions to or from the excess soluble [CPI(III)][RCu(I)] kred iron would have a negligible effect on the relevant concentrawhere k,, and k,d are the second order rate constants for the tions of the ferrous and ferric forms. The results of a reprecyano(1,lO-phenanthro1ine)iron-dependentoxidation and re- sentativetitrationare shown in Fig. 8. Each absorbance duction, respectively, of the rusticyanin, CPI(I1) and CPI(II1) spectrum in Fig. 8 was recorded after the spectrum of the represent the ferrous and ferric forms, respectively, of a rusticyanin had come to equilibrium, usually within 10 s of cyano(1,lO-phenanthro1ine)iron complex, RCu(I1) and the addition of rusticyanin to complete the reaction mixture. RCu(1) are oxidized and reduced rusticyanin, respectively, Each spectrum in Fig. 8 provided a sensitive means of quanis the equilibrium constant for the transfer tifying the fractions of the total population of rusticyanin and KRcu(,)cpI(III) of an electron from reduced rusticyanin to oxidized cy- molecules that were either oxidized or reduced. By systematano(1,lO-phenanthro1ine)iron.Equation 9 may be rearranged ically varying the ratio of sulfatoiron(I1) to sulfatoiron(III), to the redox state of the population of rusticyanin molecules was systematically manipulated to yield the standardNernst plot ( R T / F ) h ( k o x / k d= E8pr - Eku (10) shown in theinset in Fig. 8.The dataplotted inthe inset were where R is the gas constant, T i s the absolute temperature, F obtained using two different cyano(1,lO-phenanthro1ine)iron is Faraday’s constant, and E&] and E:Cu are the reduction complexes. The close correspondence between the two data potentials for the cyano(1,lO-phenanthro1ine)iron and the sets demonstrated that the identity of the catalyst had no rusticyanin, respectively. Values for the reduction potentials appreciable effect on thequantitative outcome of the results. in Table I and thekinetic constants in TableI11 were used to A value of 677 mV for the reduction potential of rusticyanin construct alinear plot according to Equation 10, as illustrated was obtained from the abscissa intercept of the Nernst plot. in Fig. 7. The reduction potential of the rusticyanin was These equilibrium experiments provided yet another indedetermined to be 680 mV from the abscissa intercept of the pendent verification of the value of the reduction potential of line in Fig. 7, a value in agreement with that reported from the rusticyanin. independent potentiometric experiments (20). DISCUSSION The reduction potential of the rusticyanin was also determined by equilibrium experiments analogous to those dePrevious kinetic studies on the electron transfer reactions scribed above in Fig. 3. The rusticyanin was introduced into between sulfatoiron and purified rusticyanin indicated that solutions of soluble iron composed of different ratios of the the rates of reaction were far tooslow to support the hypothferrous and ferric forms of sulfatoiron. A catalytic concentra- esis that rusticyanin is the primary oxidant of ferrous ions in tion of an appropriate cyano(1,lO-phenanthro1ine)iron com- the iron-dependent respiratoryelectron transport chain of T. plex was included in each mixture to facilitate rapid electron ferrooxidans (22). Indeed, the second order rate constants for
Electron Transfer between Rusticyanin and Cyano(phenanthro1ine)iron
19207
self-exchange rate constants, along with the kinetic constants in Table 111, were subsequently used to calculate 12 different apparent self-exchange rate constants for the rusticyanin. The values of these calculated constants are listed in Table 0.48 IV. Recognizing that each value in Table IV is derived from the multiplicand of five individual experimental observations, each with its own level of experimental error, the close cor20, 0.36 respondence among 10 of the 12 values in Table IV is excepB tional. One can conclude that these 10 cyano(1,lO-phenanthro1ine)iron compounds have equal kinetic access to the 2 0.24 redox center of the rusticyanin and utilize the same reaction d pathway for electron transfer. One physical interpretation of these kinetic data is that the electron transfer reactivity of 0 12 the rusticyanin is enhanced when the reaction partner possesses hydrophobic, ?r-conjugated ligands that can penetrate into the interior of the protein, thereby facilitating orbital noverlap with the protein redox center. The 4,7-diphenylsul450 600 750 fonic acid derivatives are sterically hindered from penetrating Wavelength, nm as deeply into the protein interior and demonstrate a lower FIG. 8. Absorbance spectra of rusticyanin in differentmix- reactivity with the rusticyanin. The hydrophilic sulfatoiron, tures of sulfatoiron(I1) and sulfatoiron(II1). Final concentrawhich would not be expected to penetrate the protein interior tions: msticyanin, 250 p M ; sulfate, 0.1 M; total soluble iron, 20 mM; and dicyanobis(1,lO-phenanthroline)iron,1.0 p ~ The . ratios of sul- at all, displays by far the slowest electron transfer reactivity fatoiron(II)/sulfatoiron(III) were 0.0, 1.2, 2.1, 3.0, 4.5, 8.0, 12, and w with the rusticyanin. Kinetic studies such as these are very in experiments a-h, respectively. Inset, a standard Nernstplot of the useful in defining the electron transfer reaction pathways that reductive titration of msticyanin. RCu(ZI. and RCu(I, are theconcen- are available in individual redox-active proteins. trations of rusticyanin in the oxidized and reduced states, respecA current interest of this laboratory includes the identifitively. The open and closedcircles represent data collected in the cation, isolation, and characterizationof the electron transfer presence of dicyanobis(1,lO-phenanthro1ine)iron and tetracyano(1,lO-phenanthroline)ferrate,respectively. E,$ is the totalsystem proteins responsible for aerobic respiration on soluble iron. reduction potential. The ordinate interceptand slope were determined Respiration on iron represents a principal metabolic activity incertain chemolithotrophic organisms that inhabitironby a linearregression analysis. bearing geological formations exposed to the atmosphere. Energy is derived from oxidative phosphorylation coupled to TABLE IV respiratory electron transfer. Since sulfate is the dominant Calculated self-exchange rate constantsfor the rusticyanin anionbothin the bacterium's naturalhabitatand in the Mixed ligand comdex Self-exchange rate constant laboratory culture media widely employed,the energy-yielding rnM' s" electron transfer reactions are initiated by electron donation (CN)2(phenanthroline)ziron from sulfatoiron(I1) at a standard reduction potential of, at Unsubstituted 46 its lowest, 650 mV. Preliminarystudies in this laboratory 4-Methyl 21 5-Methyl 44 suggest that exceptional diversity exists in the types of elec5-Chloro 29 tron transfer proteins that are expressed by individual mem5-Nitro 56 bers of the diverse group of microorganisms that respire on 4,7-Diphenylsulfonicacid de0.035 iron (33). An entire class of veryhigh potential electron rivative transfer proteins has begun to emerge from these studies. It (CN),(phenanthroline)iron is anticipated that the synthetic electron transfer reagents Unsubstituted 76 4-Methyl 130 characterized here will be of use in investigating the electron 5-Methyl 96 transfer reactivity of these high potential redox proteins.
:
5-Chloro 5-Nitro 4,7-Diphenylsulfonic acid derivative
66 120 0.44
~~~~~~~~~
the sulfatoiron-dependent reduction and oxidation of the rusticyanin were 2.3 and 0.74 M" s-', respectively. The sluggish electron transfer behavior of the rusticyanin with sulfatoiron may be contrasted with the relatively rapid electron transfer reactions between rusticyanin and each of the 12 organoiron compounds documented here. Some interesting features of the rusticyanin's electron transfer reactivity may be deduced from the kinetic data in Tables I1 and 111. Equations l and 2 may be combined and rearranged to yield
kxx = kxykyx/kyy
(11)
Using Equation 11, the kinetic constants in Table 11, and a value for the self-exchange rate constant of sulfatoiron of 8.7 M" s" (32), a value for theapparent self-exchange rate constant of each of the 12 cyano( 1,lO-phenanthro1ine)iron compounds listed in Table I1 was obtained. These calculated
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Electron Transfer between Rusticyanin and Cyano(phenanthro1im)iron
(1981) J. Am. Chem. SOC.103,5543-5547 13. Wherland, S., and Gray, H. B. (1977) in Biological Aspects of Inorganic Chemistry (Addison, A. W., Cullen, W., Dolphin, D., and James, B. R., eds) pp. 289-368, Wiley, New York 14. Holwerda, R. A., Wherland, S., and Gray, H. B. (1976) Annu. Rev. Biophys. Bioeng. 5, 363-396 15. Marcus, R. A,, and Sutin, N. (1985) Biochim. Biophys. Acta 811, 265-322 16. Cobley, J. G., and Haddock, B. A. (1975) FEBS Lett. 60, 29-33 17. Cox, J. C., Aasa, R., and Malmstrom, B. G. (1978) FEBS Lett. 93,157-160 18. Ingledew,W. J., Cox, J. C., and Halling, P. J. (1977) FEMS Microbiol. Lett. 2, 193-197 19. Cox, J. C., and Boxer, D. H. (1978) Biochem. J. 174,497-502 20. Ingledew, W. J., and Cobley, J. G. (1980) Biochim. Biophys. Acta 690, 141-158 21. Adman, E. T.(1985) in Metalloproteins. Part Z: Metal Proteins with Redox Roles (Harrison, P. M.,ed) pp. 1-42, Verlag Chemie, Deerfield Beach, FL 22. Blake, R. C., 11, and Shute, E. A. (1987) J . Biol. Chem. 262, 14983-14989
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23. Lewis. C. A.. Lamin. A. G.. and Indedew. W. J. (1983) . . Biochem. SOC.'Trans. 12;503 24. Lamin. A. G..Lewis.C.A.. and Ineledew. W. J. (1985) . . Znorz. k i e m . 24,1446-1450 ' 25. Schilt, A. A. (1960) J. Am. Chem. SOC.82,3000-3005 26. Sutin, N., and Gordon, B. M. (1961) J. Am. Chem. SOC.83, 7073 27. Tuovinen, 0.H., and Kelly, D. P. (1973) Arch. Microbiol. 88, 285-298 28. Ingledew, W. J. (1982) Biochim. Biophys. Acta683,89-117 29. McAndrew, R. T., Wang, S. S., and Brown, W. R. (1975) CZM Bull. 68, 101-110 30. McGinnis, J., Ingledew, W. J., and Sykes, A. G. (1986) Znorg. Chem. 25,3730-3733 31. Johnson, J. M., Halsall, H. B., and Heineman, W. R. (1983)Anal. Biochem. 133,186-189 32. Alberty, W. J., and Kreevoy, M. M.(1978)Adv. Phys.Org. Chem. 16,87-157 33. Blake, R. C., 11, Shute, E. A., and White, K. J. (1990) in Biohydrometallurgy, 1989 (Salley, J., McCready, R. G. L., and Wichlacz, P. L.,eds) pp. 391-401, Canada Centre for Mineral and Energy Technology, Canada I
supplementary Material to MIXED LIQANDWMPLEXES OP IRON WITK CYANlDB AND PHENANTHROLINE AS HEW PROBES OF llETALLOPROTEIN ELECTRON TRUlSFER REACTIVITY. AUALYSIS OF REACTIONS FROM TIIIOBACILLUB pL.BILOoXWAN8 INVOLVINGRUSTICYAUIU
Robert Blake 11, Kathy J. White. and Elizabeth A. Shute EXPERIMENTALPROCEDURES
All of the dicyanobis(l,lo-phenanth~~line)iron(II)and tetracyano(l.10p h e n a n t h r o l i n e ) f e r r a t e ( I I ) derivatives examined in the present Study were Obtained using ProCeduIes described previously for the preparation and isolation Of the mixed ligand complexes of iron(I1) with cyanide and unsubstituted 1.10- phenanthroline (25). Although the Same nethod Of preparation proved to be adequate for each substituted 1,lo-phenanthroline. the identity of the substituent on the aromatic diimine had a decided effect on the yield Of product obtained in each synthesis and in one instance necesSitated a slight modification of the generic procedure. Brief procedural details are therefore included here for the preparation of the iron(I1) coa-
a slight eodificatian (22) of a previously published procedure (191. Rusticyanin was isolated with its copper center in the oxidized state. The oxidized, purified rusticyanin was stored in 0.001 N sulfuric acid for at c without aooreciable deleterious effects. least 4 months at 4' For experiments involving oxibition of the protein, the rusticyanin vas reduced by reactinq it with an excess of sodium dithionite. The reduced c against 0.001 N sulfuric acid to remove protein was then dialyzed at '4 the excess reducing aqent. Reduced rusticyanin was renarkably stable to air oxidation. Samples Of the reduced protein Were stared in 0.001 N sulfuric was acid for UP to 3 months at 4' C before air-oxidized rusticyanin detected.
Beasurementr - Kinetic measurements Of direct electron transfer reactions Vere performed on the stopped flow Spestrophotoneter The oxidant and the reductant were prepared in described previously (22). identical solutions of 0.1 PI sulfate, pH 2.0, and added to separate syringes of the stopped flow spectrophotometer. The temperature Of the driving syringes was maintained at 25+1° C by circulating water. ROOD temperature 10 minutes in the driving solutions were allowed to equilibrate for syringes. ReactiOnE were initiated by rapidly mixing 0.1 nl of the Solution from each driving syringe. Spectral changer were linear to an absorbance of 1.8. The changes in the Oxidation state Of rusticyanin were monitored at 597 nm. The changes in the oxidation state of each orqanoiron derivative were monitored at thewavelength of maximum difference between the oxidized and reduced states Of each Compound, cis discussed below. A typical absorbance change of 2 0.06 (2-cn path length) provided acceptable signal to noise characteristics.
Kinetic experiments involving the organoiron-catalyzed electron tranafer reactions between sulfatoiron and rusticyanin were typically conducted by incorporating the appropriate catalytic amount Of the olganoiron Compound in the solution that contained the sulfatoiron in the exrrerimental DrOtOCO1 described above. Control experiments demonstrated that the order oi addition of the Catalyst had no detectable influence on the experinental Observations. meerbance
Leasure-
t
-
Absorbance spectra were obtained on a Cary On Line Instrument
14 dual-beam SDectroDhotometer rebuilt and nodified by
When used as Starting materials for the Synthesis of the corresponding tetrawano derivatives. the solid Droducts obtained above were not further purified. For purposis of spectrai and kinetic mea8UrenentSI a portion of each initial product w a ~ recrystallized from concentrated sulfuric acid. In a typical recrystallization from H S O 4 , ? g of the complex was disnolved in (95%) to give a flar;fled, bright yellow solution. Ap5-10 1 1 Of H2SOl proximately 300 m l of distilled water Were then added very s1OWly with Stlrring. Each precipitate that forned early during the dilution Underwent various Changes in hue, generally evolving to dark violet solid that Was collected, washed. and dried BE described above. The typical yield from each recrystallization vas around 8 0 % .
-
A mixture P-"ar=CJ on nf T e & & L i n e I t % r r a L s U L l of 0.5 9 of dicyanobis(l,lo-phenanthioline)i~~"(I~)and 200 a1 of an aqueous Solution containing 20 g of KCN Was tightly sealed in a Screw-Capped vessel and maintained at 100- C in a drying oven for five days. After cooling and filtration to remove the ""reacted solid, the filtrate vas extracted with portions Of Chloroform until no further violet coloration appeared in the organic layer. Evaporation of the dark orange a u e w s phase to d volume of approximately 4 0 nl with subsequent cooling to' 6 C gave a dark orange-tored crystalline product that vas collected by Suction filtration. Almost complete evaporation Of the filtrate was required before recrystallization Of each product Could be achieved from distilled water. Typical yields of the final, recrystallized tetracyano(l.10- phenanthroline)ferrat~(II) from the starting dicyanabis(l.10- phenanthroline)iron(II) ranged from 4 0 to 7 0 % .
Prenaraiion a ~ ~ ~ a n o b i s i l . l O - o h e n a n t h r o l + n e i i r o n ( I Iand ~ TBtracvanoy l O - a h e n a n t h r a l i n e l f e r r a t e i I I I 1 - Each organoiron(I1) derivative was dissolved in 0.01 N sulfuric acid and oxidized to the ferric state by the addition of a 10-fold or greater molar excess of solid lead dioiide (261. oxidation of each orqanoiron(I1) derivative Vas usually complete within 10 see after the t w o redgents had been thoPoughly mixed. The product lead sulfate and the e x c e s s lead dioxide were removed by filtration and the resulting organoiran(II1) solution was used Without further purification.
-
of Rusticvanin - Large scale growth of T. ferrooxdians (ATCC 23270) for the purification of rusticyanin was achieved by batch CUIferrous sulfate growth medium ture at ambient termeratures in the acidic described by Tuovine'n and Kelly (27) that had been supplenented with 1.6 mM cupricsulfate (22). Rusticyanin was subsequentlypufifiedto electrophoretic homogeneity from cell-free extracts of T. ferrooxldans using
m.
Ail electron transfer reactants were prepared in identical solutions of n sulfate. p H 2.0. Theeffectiveredoxpotentialofeach s u l f a t o i r o n ( ~ ~ ) / s u l r a t o i r a n ( I Imixture ~) vas calculated from the following thermodynamic diagram: 0.1
Electron Transferbetween Rusticyanin
and Cyano(phenanthro1ine)iron
thernodynanic experiments were prepared fresh daily a s a precautionary measure. Acidic solutions of the dioyanobis(l.10-phenanthroline) complexes Of iron(I1I) Yere considerably lees stable than those Of the corresponding tetracyano derivatives. Consequently, experimental Observations on the kinetic or thermodynamicpropertiesofthedicyanobis(1,lop h e n a n t h r a 1 i n e ) i I o n ~ I I I ) com~lexeswere limited to solutions utilized within 90 nin Of their preparation.
19209
TaLBLE 111
Second order rate Constants for electron transfer between cyana(l,lophenanthro1ine)iron and rusticyanin
Rate constant for Rate constant Mixed ligand RESULTS
Complex
TlsLE I
CDnPleX
Absorbance maximum of reduced species. nn
Reduction
cred,
unrubatituted
0.10
4.1
4-methyl
0.20
5.6
5-methyl
0.12
potential, mv
nn-lcm"
46 (CN)2(phen12iron Unsubstituted
514
4.65
800
4-methyl
507
4.13
765
5-methyl
515
3.76
790
522
4.40
855
5-ChlorO
pM-l
s-1
5-nitro
536
5.26
885
bathos
537
4.42
755
unsubstituted
450
1.85
570
4-methyl
463
1.64
540
5-methyl
457
1.50
565
5-chloro
480
1.75
660
5-nitro
521
2.09
680
Dathaa
536
1.76
525
5-chloro
0.022
5-nitro
0.019
batho'
0.073
6.8
24
1.2
(CN)4(phen)iron
6.2
(CN)4(phen)iron
a
*-I
""1
for Oxidation Of RCu(1)
(CN)2(phen)2iron
SpeFtral and electrochemical properties Of cyano(l,lO-phenanth=oline)iron compounds
nixed ligand
reduction Of RCu(I1)
a
unsubstituted
55
0.67
4-methyl
33
0.39
5-methyl
42
0.58
5-chloro
7.4
1.6
5-nitro
5.4
bathos
6.3
0.018
4,7-diphenylsulfonic acid
'i
4.7-diphenylsulfonic acid
TABLE I1
Second Order rate Constants for electron transfer between soluble cyano(l.lo-phenanthroline)iron
Rate constant Mixed ligand
for
Oxidation by F e ( I I 1 ) m-l
complex
s-l
Rate constant
iron and
YI
for
reduction by Fe(I1) nn-l s-l
(CN)2(phen)2iron Unsubstituted
1.2
60
4-methyl
2.1
22
5-methyl
1.4
40
5-chloro
0.51
120
5-nitro
0.24
240
bath#
16
89
(CN)4(phen)iron
a
unsubstituted
1200
4-methyl
2700
1.8
5-methyl
890
5.0
5-chloro
250
29
5-nitro
130
37
bathaa
5900
4.7-diphenyl5ulfanic
acid
5.5
4.4
:I 00
Felm), mM
Electron Transfer between Rusticyanin
19210
and Cyano(phenunthro1ine)iron
0.06 IC CJ, Lo
5 0.04 C
0
e2 0.02 n
a Q
-5.0
0
20
10
30
O
40
0.75
0.50 0.25
Time, ms
1.00
Time, s
Pig. I . Time course Of the ObsOrbanCe changes at 597 nm when oxidised rustisyanin was mixed with different cmcentzations of t ~ t = . ~ y 0 i n o ( 5 - n i t ~ ~ - l , ~ a ph~n~nthrolin~~ferrat~l11 Final 1. concentrations after mixing were: rusticyanin. 2 1 )in: s u l f a t e ,0 . 1 M: and t e t = a c y a n 0 ( 5 - n i t T O - 1 , 1 0 phenanthroline)ferrate(II), 100. 6 7 , and 3 0 pM in experiments a, b, and f, respectively. The time-dependent absorbance changes Yere sufficiently rapid to permit only about 2 5 , 50, and 7 5 % Of the anticipated changes in absorbance to actually be observed after flow was Stopped in experiments a. b, and E, respectively. Since all three experimental traces exhibited pseudofirst order Linetic behavior. the time course Of each reaction that Occurred before the cesSatiDn of flaw was reconstructed and is represented by the M portion of each curve in the figure. All three extrapolated curves intersected at a point consistent With the total change in absorbance anticipated far the reaction. A Value of 2 . 0 t 0.3 msec was determined for the deadtime of the stopped flaw spectrophotometer using data such as those included herein.
0.08
I
6 0.06
I
I
0.50
0.75
B
lc
CJ,
Lo
al
2
0.04
0
e 1
1: 0 . 0 2 a
a 0 0 I 100 2
r
a,-
that describes the .protein reduction reaction. tine course br the ~~=d, tetracyano(5-chloro- l , l o - p h e n a n t h r o l i n e ) f e = ~ ~ t = - ~ ~ t = lfe(II1)-dependent oxidation of the ruaticyanin. The final concentrations after mixing were: sulfate, 0 . 1 M: ~e(111). 1.0 mM: rusticyanin, 15 on: and tetracyano(5chlaro-l,lO-phenanthroline)ferrate. 0.71 uM. -, a l i n e a r plot according to Equation 8,beloY. Of the integrated Steady-state rate equation that describes the protein oxidation reaction.
20
20
40
Iron
60
00
100
Cornplex,uM
nitro-l,lo- p h e n a n t h r o l i n e l f e r r a t e ( I 1 ) : b . dicyanobis(4-methyl-1,lOphenanthroline)iron(II): and P , dlcyanobis(l.10- phenanthroline)iron(II). 8, oxidizingagents Were: a. d i c y a n o b i s ( 4 . 7 - d i p h e n l y s u l f o n a t e - 1 , l O phenanthroline)iron(Ill]: tetracyana(l,lo-phenanthroline)ferrate(IIr); and 2 , t e t ~ a c y a n o ( 4 - m e t h y l - 1 . 1 0 - p h e n a n t h r o l i n e ) f ~ ~ ~ ~ t ~ ~ l l l l .
L?.
1.00
Time, s
1i i
0.25
Electron Transfer between Rusticyanin and Cyano(phenanthro1ine)iron -
Steadv-state s eTrqheueaOeecfnatcciehno n s catalytic cycle of the cyano(l,lo-phenanthroline)iron-cataly.ed, Fe(1I)dependent reduction Of the rusticyanin is illustrated by the following mechanism:
where RCu(I1) and ncu(1) are oxidized and reduced rusticymin, respectively. Fig. 68 s h o w a kinetic trace of the Fe(I1)- dependent. tetracyano(l.10phenanth~oline)fe==.te-catalyred reductionOfrusticyanin,while Fig. 68 tetrscyano(5-chloro-1.10shows an example Of the Fe(II1)-dependent, phenanthrolinelferrate-catalyzed oxidation Of rusticyanin. Neither of the kinetic traces in Fig. 6 could be described mathematically ae a single exponential function Of time. Instead, each time-dependent change in absorbance at 597 nm was analyzed using a classical Michaelis-Menten kinetic treatment. The sequence of events in Equation 4 may be seen as a double displacement ping Pong Bi Bi Kinetic mechanism where the cyana(l.10phenanthro1ine)iron derivative cycles between the midized and the reduced states. Applying the usual steady-state approximation to the concentration Of the catalyst in the example given in Equation 4, it is evident that i~Yanall,lO-phenanthraline)ironj
d Rcu(1I) d t
-
1
k ~ e [ IFe(II1 ~ ~ ) I
+
where V
=
19211
k F e ~ I I ~ I F e ~ I I l ] I c y a n o ( l , ~ O - p h e n a n t h r o l i n e ) i r a n Kj . =
kF.(II~IFe~IIll/kred, AAt is the absorbance at time t minus thatat the end Of the reaction (At-A-I, and AAm is the total absorbance change Observed Selecteddata spanned 8 0 1 Of the total absorbance pointsthat lAo-Am). change in Fig. 68 were used to Construct the linear plot according to Equation 6 Shown in the to the figure. A value for X was determined from either the slope or the abscissa intercept of this linear plot. A corresponding value for kred vas then readily calculated from this value of K using the appropriate value for kF,(,,) in Table 11. If the cyano(l,lO-phenanthroline)iron Were enplayed to Catalyze the FelII1)-dependent oxidation of the rusticyanin. the equivalent to Equation 5 is (71 [cyano(l,lo-phenanthroline)ironl
d RCuIIl d t
-
1
kFe(lII) [Fe(lIIlI
+--"
1
koXIRCu(Il i
1 (51
kredlRCu(IIll
Where kFe(IIl and kred are the second order rate constants for the F e ( I I ) dependent reduction O f t h e c y a n o ~ l , l o - p h e n a n t h r o l i n e ) i r o n o and the c y a n o ( l , l o - p h e n a n t h r o l i n e ) i r o n o - d e p e n d ~ ~ t reduction of the rusticyanin, respectively. The lack of any evidence for precursor complex fornation in any of the stopped flow Spectrophotometric experiments discussed above eliminated the usual collection of first order rate constants in Equation 5 that would normally define the maximum velocity of the catalyzed reaction. If the change in the Concentration of Fe(II1 is assumed to be negligibly m a l l during the course Of the reaction, then Equation 5 nay be rearranged and integrated over time and oxidized rusticyanin concentrations to yield
Where kFelIIl) and kox are the second order rate constants for the Fe1III)dependent oxidation of the ayano(1,lO-phenanthroline)iron(Il) and the c y a n o ( l , l o - p h e n a n t h r o l i n e J i r o n o - d e p e ~ d ~ ~oxidation t of the rusticyanin, respectively. If the change in the concentration Of Fe(II1) is assumed to be negligibly small. Equation 7 may be rearranged and integrated Over the usual limits to yield
