Mixed Ligand Complexes of Iron with Cyanide and Phenanthroline as New Probes of Metalloprotein Electron Transfer Reactivity ANALYSIS OF REACTIONS INVOLVING RUSTICYANIN FROM THIOBACILLUS FERROOXIDANS*

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 were determined and spanned from 500 to 900 mV. The one-electron transfer reactions between each of these substitution-inert reagents and the high potential blue copper protein, rusticyanin, from Thiobacillus ferrooxidans were studied by stopped flow spectropho-tometry 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. of and small organometallic complexes with isolated electron transport proteins, the cytochromes (1-4), type I blue copper proteins (5-8), and various iron

Extensive kinetic data are available on the electron transfer reactions of selected inorganic and small organometallic complexes with isolated electron transport proteins, especially the cytochromes (1)(2)(3)(4), type I blue copper proteins (5)(6)(7)(8), and various iron sulfur proteins (9)(10)(11)(12). One goal of such research has been the elucidation of reaction pathways available to the proteins and the manner in which the polypeptide envelopes have modified the inherent reactivity of the metal center. In particular, the Marcus theory of electron transfer has been widely employed to deduce, from the relevant kinetic and thermodynamic data, the factors governing the rates of outer sphere electron transfer reactions between small organometallic complexes and metalloproteins (13)(14)(15), and references therein). A wide assortment of well characterized outer sphere electron transfer reagents varying in charge, reduction potential, and ligand structure are available to systematically probe the reactive sites of metalloproteins that have reduction potentials in the approximate range of 0-400 mV.
Rusticyanin is a 16.3-kilodalton, type I blue copper protein *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. isolated from Thiobacillus ferrooxidans, an acidophilic chemolithotroph that grows autotrophically on ferrous ions (16)(17)(18)(19). The protein is thought to be expressed into the periplasmic space of this Gram-negative bacterium and to play an important, although as yet undefined, role in the iron respiratory electron 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 to pH 0.2; and its reduction potential is 680 mV (20), a value 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 studies on the electron transfer properties of purified rusticyanin utilized sulfatoiron, an inorganic complex of physiological significance to the intact organism (22-24). A principal barrier to further probes of the rusticyanin's electron transfer reactivity is that relatively few well characterized inorganic or small organometallic complexes are available with the appropriate electrochemical, solubility, and structural properties that are required 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 introducing different substituents on the aromatic diimine and varying the stoichiometry of the substituted phenanthroline in the final organoiron complex, a series of substitution-inert electron transfer reagents were constructed with reduction potentials from 500 to 900 mV. Kinetic and thermodynamic studies on the electron transfer reactions of each of these substitution-inert iron complexes with both sulfatoiron and rusticyanin are presented. The family of electron transfer reagents described herein could prove useful in probing the electron transfer reactivity of this novel class of high potential respiratory proteins.

EXPERIMENTAL PROCEDURES AND RESULTS'
Absorbance Properties-Both the redox potential and the electron transfer reactivity of the ferrous/ferric couple are profoundly influenced by the ligands coordinated to the iron cation. Hexoaquoiron has a reduction potential of 770 mV and reacts relatively slowly in its electron transfer reactions both with itself (self-exchange electron transfer) and with other electron transfer reagents. The conjugate base of hydro-' 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. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. 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 much lower reduction potential, 410 mV, than does hexoaquoiron. Conversely, the aromatic diimine 1,lO-phenanthroline binds much more strongly to the ferrous ion than it does to ferric ions, with the consequence that tris(1,lO-phenanthro1ine)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 in Fig. 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 in the complex. Curves a, b, and c represent cyano(1,lOphenanthroline)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(lJ0-phenanthro1ine)iron complexes were obtained 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-phenanthro-1ine)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)iron complex 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- 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,lO-phenanthro1ine)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 could be 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)iron complex 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 the concentration 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 constants for 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 the soluble iron. Values of these second order rate constants were obtained for both the Fe(II1)-dependent oxidation and the Fe(I1)-dependent reduction of each of the 12 cyano(1,lO-phenanthro1ine)iron compounds investigated and are summarized in Table I1 (Miniprint).
The kinetic data summarized in Table I1 were exploited to estimate the reduction potential of each cyano(1,lO-phe-nanthro1ine)iron complex. Each substitution-inert organoiron complex featured in Table I1 would be expected to transfer an electron by an outer sphere bimolecular 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 the kinetic and thermodynamic properties of the reaction partners may frequently be correlated by the "cross-reaction" equation developed by Marcus (13-15): kxv = Jkxx k w KXY (1) where kxy is the second order rate constant for the transfer of an electron from X to Y, kxx and k y y are the two selfexchange rate constants (e.g. for the transfer of an electron from one molecule of X to another molecule of X), and KXY is the equilibrium constant for the electron transfer. Each self-exchange rate constant is a measure of the intrinsic reactivity of each reactant and is related to the energy 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 outer sphere electron transfer reactions, but it 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 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 that KXy = l/Kyx, Equation 1 may be divided by Equation 2 and the dividend rearranged to yield 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 I T were used in accordance with  Equation 3 to calculate an equilibrium constant for the electron transfer between sulfatoiron and each cyano(1,lO-phen-anthro1ine)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 to be 710 mV (22). Using this potential of the sulfatoiron as a point of reference, the reduction potential of each cyano(1,lO-phenanthro1ine)iron compound in Table I1 was then determined. The value of each reduction potential determined from the kinetic studies described above was then verified by independent equilibrium experiments that employed soluble iron and the cyano(1,lO-phenanthro1ine)iron complex of interest. A representative example of such an equilibrium experiment is illustrated in Fig. 3. Each absorbance spectrum in Fig. 3 was taken after a 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 that any net electron transfer reactions to or from the limiting dicyanobis( 1,lO-phenanthro1ine)iron would 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)iron molecules 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-phenanthro-1ine)iron molecules was systematically manipulated to yield the standard Nernst plot shown in the inset in Fig. 3. A value of 800 mV for the reduction potential of dicyanobis(1,lO-phenanthro1ine)iron was obtained from the abscissa intercept of the Nernst plot. Similar equilibrium experiments were performed on each of the 12 cyanobis(1,lO-phenanthro-1ine)iron compounds investigated. Acceptable agreement (within 5-15 mV) was observed between the value of each 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-phenanthro-1ine)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-phenanthro-1ine)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 in a 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 a single 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 data represent 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 rate constants for each electron transfer reaction were directly proportional to the concentration 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-phenanthro-1ine)iron complexes were too rapid to be accurately investigated by direct mixing experiments in the stopped flow spectrophotometer. Noting that the bimolecular 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-phenanthro-1ine)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 the approach 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 measurements and the corresponding values determined by the indirect steady-state kinetic approach outlined in the Miniprint. The latter steady-state approach was 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 constants obtained by the steady-state approach are included in Table 111.
The kinetic data summarized in Table I11 were  where k,, and k,d are the second order rate constants for the cyano(1,lO-phenanthro1ine)iron-dependent oxidation and reduction, respectively, of the rusticyanin, CPI(I1) and CPI(II1) represent the ferrous and ferric forms, respectively, of a cyano(1,lO-phenanthro1ine)iron complex, RCu(I1) and RCu(1) are oxidized and reduced rusticyanin, respectively, and KRcu(,)cpI(III) is the equilibrium constant for the transfer of an electron from reduced rusticyanin to oxidized cyano( 1,lO-phenanthro1ine)iron. Equation 9 may be rearranged to where R is the gas constant, T i s the absolute temperature, F is Faraday's constant, and E&] and E: Cu are the reduction potentials for the cyano(1,lO-phenanthro1ine)iron and the rusticyanin, respectively. Values for the reduction potentials in Table I and the kinetic constants in Table I11 were used to construct a linear plot according to Equation 10, as illustrated in Fig. 7. The reduction potential of the rusticyanin was determined to be 680 mV from the abscissa intercept of the line in Fig. 7, a value in agreement with that reported from independent potentiometric experiments (20).
The reduction potential of the rusticyanin was also determined by equilibrium experiments analogous to those described above in Fig. 3. The rusticyanin was introduced into solutions of soluble iron composed of different ratios of the ferrous and ferric forms of sulfatoiron. A catalytic concentration of an appropriate cyano(1,lO-phenanthro1ine)iron complex was included in each mixture to facilitate rapid electron  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 net electron transfer reactions to or from the excess soluble iron would have a negligible effect on the relevant concentrations of the ferrous and ferric forms. The results of a representative titration are shown in Fig. 8. Each absorbance spectrum in Fig. 8 was recorded after the spectrum of the rusticyanin had come to equilibrium, usually within 10 s of the addition of rusticyanin to complete the reaction mixture.
Each spectrum in Fig. 8 provided a sensitive means of quantifying the fractions of the total population of rusticyanin molecules that were either oxidized or reduced. By systematically varying the ratio of sulfatoiron(I1) to sulfatoiron(III), the redox state of the population of rusticyanin molecules was systematically manipulated to yield the standard Nernst plot shown in the inset in Fig. 8. The data plotted in the inset were obtained using two different cyano(1,lO-phenanthro1ine)iron complexes. The close correspondence between the two data sets demonstrated that the identity of the catalyst had no appreciable effect on the quantitative outcome of the results. A value of 677 mV for the reduction potential of rusticyanin was obtained from the abscissa intercept of the Nernst plot. These equilibrium experiments provided yet another independent verification of the value of the reduction potential of the rusticyanin.

DISCUSSION
Previous kinetic studies on the electron transfer reactions between sulfatoiron and purified rusticyanin indicated that the rates of reaction were far too slow to support the hypothesis that rusticyanin is the primary oxidant of ferrous ions in the iron-dependent respiratory electron transport chain of T. ferrooxidans (22). Indeed, the second order rate constants for    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 the apparent self-exchange rate constant of each of the 12 cyano( 1,lO-phenanthro1ine)iron compounds listed in Table I1 was obtained. These calculated 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   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 correspondence among 10 of the 12 values in Table IV is exceptional. One can conclude that these 10 cyano(1,lO-phenan-thro1ine)iron compounds have equal kinetic access to the redox center of the rusticyanin and utilize the same reaction pathway for electron transfer. One physical interpretation of these kinetic data is that the electron transfer reactivity of 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 overlap with the protein redox center. The 4,7-diphenylsulfonic acid derivatives are sterically hindered from penetrating as deeply into the protein interior and demonstrate a lower reactivity with the rusticyanin. The hydrophilic sulfatoiron, which would not be expected to penetrate the protein interior at all, displays by far the slowest electron transfer reactivity with the rusticyanin. Kinetic studies such as these are very useful in defining the electron transfer reaction pathways that are available in individual redox-active proteins.

(CN)2(phenanthroline)ziron
A current interest of this laboratory includes the identification, isolation, and characterization of the electron transfer proteins responsible for aerobic respiration on soluble iron. Respiration on iron represents a principal metabolic activity in certain chemolithotrophic organisms that inhabit ironbearing geological formations exposed to the atmosphere. Energy is derived from oxidative phosphorylation coupled to respiratory electron transfer. Since sulfate is the dominant anion both in the bacterium's natural habitat and in the laboratory culture media widely employed, the energy-yielding electron transfer reactions are initiated by electron donation from sulfatoiron(I1) at a standard reduction potential of, at its lowest, 650 mV. Preliminary studies in this laboratory suggest that exceptional diversity exists in the types of electron transfer proteins that are expressed by individual members of the diverse group of microorganisms that respire on iron (33). An entire class of very high potential electron transfer proteins has begun to emerge from these studies. It is anticipated that the synthetic electron transfer reagents characterized here will be of use in investigating the electron transfer reactivity of these high potential redox proteins.   Robert Blake 11, Kathy J. White. and Elizabeth A. Shute

EXPERIMENTAL PROCEDURES
All of the dicyanobis(l,lo-phenanth~~line)iron(II) and tetracyano(l.10phenanthroline)ferrate(II) 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 untion proved to be adequate for each substituted 1,lo-phenanthroline. the substituted 1.10-phenanthroline (25). Although the Same nethod Of preparaidentity of the substituent on the aromatic diimine had a decided effect on the yield Of product obtained in each synthesis and in one instance necesdetails are therefore included here for the preparation of the iron(I1) coa-Sitated a slight modification of the generic procedure. Brief procedural tetrawano derivatives. the solid Droducts obtained above were not further When used as Starting materials for the Synthesis of the corresponding 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 proximately 300 ml of distilled water Were then added very s1OWly with Stlr-5-10 1 1 Of H2SOl (95%) to give a flar;fled, bright yellow solution. Ap-

ring.
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 % . Reduced rusticyanin was renarkably stable to air oxidation.

P-"ar=CJ
Samples Of the reduced protein Were stared in 0.001 N sulfuric acid for UP to 3 months at 4' C before air-oxidized rusticyanin L?. t e t r a c y a n a ( l , l o -p h e n a n t h r o l i n e ) f e r r a t e ( I I r ) ; and 2, tet~acyano(4-methyl-1.10-phenanthroline)f~~~~t~~llll.

Steadv-state
-The sequence Of reactions in each catalytic cycle of 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 -c a t a l y . e d , 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. phenanth~oline)fe==.te-catalyred reduction Of rusticyanin, while w h e r e V = 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 j .

kF.(II~IFe~IIll/kred, AAt is the absorbance at time t minus that at the end Of the reaction (At-A-I, and
AAm is the total absorbance change Observed change in Fig. 68 were used to Construct the linear plot according to Equa-lAo-Am). Selected data points that spanned 8 0 1 Of the total absorbance tion 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.

FelII1)-dependent oxidation of the rusticyanin. the equivalent to Equation 5
If 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 cyano(l,lo-phenanthrolineJirono-depe~d~~t oxidation 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