J . Phys. Chem. 1991, 95,6034-6040
6034
Therefore, the reduced kh, for the compartmentalized systems may be qualitatively explained in terms of a combined effect of the microenvironmental polarity and the sterically hindered loose ion-pair state. The values of kd should depend on the local concentration of bound MVZ+around the Py site. At a higher local concentration of MV2+the rate of an electron relay from MV'+ to the nearest MV2+dication is faster.29 Among the hydrophobic groups employed, the lauryl group exhibited the largest kd,suggesting that MV2+ can penetrate into the lauryl aggregate so as to increase the local concentration of MV2+ around the Py site. It is reasonable to consider that kb,2is mainly determined by and (MV'+) formed as a result of the the distance between electron relay (step 6 ) . Thus, kb,2may also depend on the local concentration of MV2+. At a lower acceptor concentration a single-step electron relay from MV'+ to (MV2+) shown in step 5 will create a longer separation between and (MV'+), leading to a reduced kb,2.A larger kb,2 observed for the lauryl terpolymer suggests a higher MV2+ local density around the Py site.
w+
w+
Conclusion The Py chromophore was compartmentalized in the hydrophobic aggregate of the amphiphilic polyelectrolytes (Chart I)
in aqueous solution. The added MV2+ was concentrated on the surface of the hydrophobic aggregate. However, the compartmentalized Py moiety was sterically protected from a close contact with bound MV2+. The rate of CR of the primary ion pair was slowed by an order of magnitude, while very fast photoinduced forward ET occurred (kET> 10" s-l), thus giving rise to charge separation that persisted for hundreds of microseconds. In sharp contrast, the reference polymer, in which the Py residue is not compartmentalized, showed a rapid CR with a rate constant of the order of 10" s-I. The reduced CR rate for the compartmentalized system was qualitatively explained in terms of a sterically hindered loose structure for the primary ion pair. The lauryl group was significantly less effective in compartmentalizing the pyrene moiety than the cyclododecyl and adamantyl groups. An optimal compartmentalization of the chromophore may be achieved by a judicious choice of the hydrophobic group, which will lead to an optimal efficiency for charge separation. Registry No. (A)(La)(Py) (copolymer), 134131-50-3;(A)(Cd)(Py) (copolymer), 134110-08-0 (A)(Ad)(Py) (copolymer), 13411049-1; PyMAm, 134110-06-8;CdMAm, 13675-35-9;AdMAm, 134110-07-9; 1pyrenecarboxaldehyde,3029- 19-4; 1-pyrenylmethylamine hydrochloride, 93324-65-3;2-adamantylamine, 13074-39-0; methylviologen, 1910-42-5; ammonia, 7664-41-7; methacryloyl chloride, 920-46-7; cyclododecylamine, 1502-03-0.
Electrochemical Study of Kinetics of Electron Transfer between Synthetic Electron Acceptors and Reduced Moiybdoheme Protein Sulfite Oxidase L. A. Coury, Jr.; Royce W. Murray,* Kenan Laboratories of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290
J. L. Johnson, and K. V. Rajagopalan Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 2771 0 (Received: February 1 1 , 1991)
The electrocatalytic oxidation of sulfite using transition-metal complex or cytochrome c mediators and the molybdoheme protein sulfite oxidase (SO) as catalysts is used to measure the rate constant (k12)for cross electron transfer between mediator and enzyme. The enzyme heme is the reaction site for cobalt phenanthroline complex mediators; the Mo fragment obtained by tryptic cleavage of native enzyme is unable to reduce the Co species. Within a structurally similar set of cobalt complexes, when (mediator SO) A E O ' < 0.2 V, k I 2varies with reaction free energy according to relative Marcus-Hush theory; we find k l l = 1 X lo8 M-I s-I for the apparent electron self-exchange rate constant of the enzyme heme site. Attempts to achieve faster heme turnover rates result in a shift of rate control to another process, possibly the internal Mo Fe electron transfer. Ionic strength effects indicate a negative charge at the heme site and substantial electrostatic rate effects.
-
Electron self-exchange rate constants (kll) for redox proteins can be derived from NMR and EPR measurements' on mixtures of oxidized and reduced Drotein, and from ~ross-electron-transfer2~ reaction rates with small-moleculereagents! These studies include "blue" cop r proteins,' flavodoxin,5 high-potential iron proteins (HiPIPs),p"and several cytochromes c . ~ , ~The ~ . relationships ~ between reaction free energy and intramolecular electron-transfer rates between protein redox sites and protein-attached synthetic redox sites,' and in protein/protein complexes? have also been intensively investigated in recent years. In the case of the molybdoheme protein sulfite oxidase (SO), prior reports9J0 contain no information on reaction free energy effects or enzyme self-exchange dynamics. Elucidation of the electron-transfer dynamics of biomolecules like SO is relevant to *Corresponding author. 'Present address: Department of Chemistry, Duke University, Durham, NC 27706. 0022-3654/91/2095-6034$02.50/0
understanding the complexities of metabolic redox pathways;" appreciating the reactivity of enzymes like SO is also of interest (1) (a) Dixon, D. W.; Hong, X.;Wochler, S.E.;Mauk, A. G.; Sishta, B. P.J . Am. Chem. Soc. 1990,112,1082-1088, and referenccs cited thmin. (b) Grocntveld, C. M.;Canters, G. W. J. Biol. Chem. 190,263, 167-173. (c) Dahlin, S.;Reinhammar, B.; Wilson, M.T. Blochem. J . 1984,218,609-614. (2) (a) Marcus, R. A. Annu. Reo. Phys. Chem. 1964,15, 155-196. (b) Marcus, R. A.; Sutin, N.Biochim. Biophys. Acta 1985,811,265-322. (3) Wherland, S.; Gray, H. B. In Biological Aspecrs ofInorgank Chemistry; Addison, A. W.,Cullen, W. R.,Dolphin, D., James, B. R., Eds.; Wiley: New York, 1977; pp 289-368. (4) (a) McArdle, J. V.; Coylc, C. L.; Gray, H. B.; Yoneda, G. S.;Holwerda, R. A. J . Am. Chem. Soc. 1977,99,2483-2489. (b) McArdle, J. V.; Gray, H. B.; Crcutz, C.; Sutin, N. J . Am. Chem. Soc. 1974,96,5737-5741. ( 5 ) (a) Simondsen,R.; Tollin, G. Biochemistry 1983,22. 3008-3016. (b) Meyer, T. E.;Rzysiecki, C. T.; Watkins, J. A,; Bhattacharyya, A,; Simondscn, R. P.; Cusanovich, M. A.; Tollin, G. Proc. Natl. Acad. Sci. 1983, 80, 6740-6744. (c) Cusanovich, M.A.; Meyer, T. E.;Tollin, G . Ado. Inorg.
Biochem. 1988, 7, 37-91.
0 1991 American Chemical Society
The Journal of Physical Chemistry, Vol. 95, No. 15, 1991 6035
Electrocatalytic Oxidation of Sulfite because Mo is the only second-row transition metal essential for life.I2 This paper presents a Marcus-Hush2 analysis of the free energy-rate relationship for intermolecular electron transfer (k12) between sulfite oxidase and a variety of mediator oxidant species, from which we obtain the first estimate of the a parent electron self-exchange rate constant (kll) for the SO Fen'/ I heme site. The kI2rate measurements are based on electrocatalytic currentsI0 for the regeneration of oxidized mediator species (ox) which are consumed by enzyme and a saturating excess of sulfite in the series of reactions
P
red - leox
-
+ Enz(FeI1,MoIV)
kll
-
Enz( Felll,MoIV) ox
ox
+ Enz(FelI,MoV)-L!
-
Enz(FelI1,MoV) Enz(FelI,MoV1)+
+ H20
red
(1)
+ Enz(FelI1,MoIV)
Enz( FelI,MoV) red
Enz(Fe1I,MoV1)
-
IjT B
(2)
(3)
+ Enz(FelI1,MoV)
F
A pA/cme
(4)
(5)
+
Enz(Fe1l,MoIv) + Sod2- 2H+ ( 6 )
where ox and red represent the mediator couple, the ox form of which oxidizes the SO heme Fe" site with rate constant klz. The product Fell1site then undergoes intramolecular electron transfer to form the MoV1active state that consumes sulfite. The pattern of (2)-(6) has been established in the 1iterat~re.I~From anaerobic electrocatalyticstudies'& of [ C ~ ( b p y ) ~as ] ~mediator + and analysis of the dependencies of current on enzyme and mediator concentrations, we have established that under selected conditions the rate-determining steps are (2) and (4), whose rate constants we assume are equal. In the present experiments, the electrocatalytic scheme is employed to determine kI2 for a number of different mediators, including a structurally similar set of cobalt phenanthrolines and cytochrome c. At constant ionic strength, values of k12for the cobalt complexes vary with their reaction free energies with the for enzyme in the manner predicted by Marcus-Hush the0ry~9~ the slower reacting complexes; an apparent k l , for the enzyme emerges from this comparison. Changes in ionic strength provoke substantial change in kI2and reveal a considerable electrostatic (6)Wherland, S.;Gray, H. B. Proc. Natl. Acad. Sci. U.S.A. 1976,73, 2950-2954. (7)(a) Bowler, B. E.; Meade, T. J.; Mayo, S.L.; Richards, J. H.; Gray, H. B. J. Am. Chem. Soc. 1989,I l l , 8757-8759. (b) Meade, T. J.; Gray, H. B.; Winkler. J. R. J. Am. Chem. Soc. 1989,111,4353-4356.(c) Mayo, S. L.; Ellis, W. R.; Crutchley, R. J.; Gray, H. B. Science 1986,233,948-952. (d) Axup, A. W.; Albin, M.; Mayo, S.L.; Crutchley, R. J.; Gray, H. B. J . Am. Chem. Soc. 1988, 110,435-439.(e) Karas, J. L.; Lieber, C. M.; Gray, H. B. J . Am. Chem. Soc. 1988, 110,599600. (f) Bcchtold, R.; Gardineer, M. B.; Kazmi, A.; van Hemelryck, B.; Isied, S.S . J . Phys. Chem. 1986,90, 3800-3804. (g) Isied, S.S.;Vassilian, A.; Magnuson, R. H.; Schwarz, H. A. J . Am. Chem. Soc. 1985, 107, 7432-7438. (8)(a) McLendon, G.; Miller, J. R. J . Am. Chem. SOC. 1985, 107. 781 1-7816. (b) Conklin, K. T.; McLendon, G. J . Am. Chem. Soc. 1988,110, 3345-3350. (c) McLendon, G.; Miller, J. R. J . Am. Chem. SOC.1985,107, 781 1-7816. (9)(a) Kipke, C. A.; Cusanovich, M. A,; Tollin, G.;Sunde, R. A.; Enemark, J. H. Biochemistry 1988,27,2918-2926. (b) Barker, P. D.; Hill, H. A. 0.In Oxidases and Related Redox Systems; King, T. E., Mason, H. S., Morrison. M., Eds.; A. R. Liss, Inc.: New York, 1988;pp 419-433. (IO) (a) Coury, L. A.; Oliver, B. N.; Egekeze, J. 0.; Sosnoff, C. S.; Brumfield,J. C.; Buck, R. P.; Murray, R. W. Anal. Chem. 1990,62,452-458. (b) Oliver, B. N.; Coury, L. A,; Egekeze. J. 0.; Sosnoff, C. S.;Zhang, Y.; Murray, R. W.; Keller, C.; Umana, M. X . In Biosensor Technology, Buck, R. P.. Hatfield. W. E.. Umalla.. M... Bowden.. E. F... Eds.:. Dekker: New York. 19901 pp i17-i3s. . (11)Scott, R. A.; Mauk, A. G.; Gray, H. B. J . Chcm. Educ. 1985,62, 932-938. (12)Hay, R. W. Eio-Inorganic Chemistry; Ellis Horwood: Chichester, UK, 1987;p 19. (13) Rajagopalan, K. V. I n Molybdenum and Molybdenum-Conrainlng Enzymes; Coughlan, M., Ed.; Pergamon: Oxford, UK, 1980; pp 241-269.
+.lo
E/!// ' 4 . 2
Figure 1. Cyclic voltammograms at 0.0303 cmz EFT3 working electrode, 2 mV/s. (A) Background scan in 20 mM TRIS buffer, pH 7.45,O.l M KCI; (B) 0.55 mM Na2S03 added; (C) Upper curve: 44 pM [Cohen)^]^+; 0.53 mM Lower curve: 43 pM [ C o ( p h ~ n ) ~ ] ~1.+31, pM rat liver SO, 0.52 m M sulfite added. Cross represents 0 V vs Ag/ AgCl and zero current.
component in the reaction. In faster mediator/SO reactions, brought about by (i) more oxidizing versions of the cobalt complexes, (ii) low ionic strength, or (iii) mediators with very large kZ2values, the cross reaction k12rate constants attain a common, apparently maximal value which may represent a shift of heme turnover rate control to the internal Mo Fe electron transfer, reactions 3 and 5 .
-
Experimental Section Chicken and rat liver sulfite oxidase samples [1.8.2.1], isolated as previously described,14were stored in 1-mL quantities at 0 OC. The ferricyanide reductase activity of thawed batches of whole enzyme was spectrophotometrically assayed" in pH 8.5,O.l M TRIS/ 100 pM EDTA (TRIS = tris(hydroxymethy1)aminomethane), using initial rate measurements and conditions pseudo first order in ferricyanide (Le., a kinetically inexhaustible supply of reduced enzyme maintained by a lo6 molar ratio of sulfite). The mean second-order rate constant for reduction of ferricyanide by different batches of chicken liver enzyme thawed over a 5month interval is (7 f 2) X IO5 M-' s-l. Similarly, k I 2 is (9 f 1) X lo5 M-l s-l for rat liver enzyme over a 7-month interval. Rat enzyme was cleaved with trypsin and isolated as the Mo fragment as reported elsewhere.'" The fragment concentration was determined spectrophotometrically at 350 nm (e = 5 M-' cm-I).16 Unless otherwise noted, solutions for electrochemistry were Ar-saturated 0.02 M TRIS/O.l M KCI, adjusted to pH 7.5 with HCl or KOH. The cytochrome c employed (Sigma type VI, horse heart, prepared without trichloroacetic acid, as received) is electroactive at pyrolytic graphite electrodes under these solution conditions.Iq Cobalt(I1) complexes were prepared in situ by injecting an appropriate volume of degassed, aqueous CoCI2solution via a septum cap into an Ar-flushed vial containing a 4-fold molar ratio of bpy (2,2'-bipyridine) or phen (1,lO-phenanthroline) ligand, or a 3-fold ratio of terpy (2,2',2"-terpyridine). Ligands (G. F. Smith Chemicals, Aldrich, Alfa, PolySciences) were used as received. Equipment for cyclic voltammetry (CV) and chronoamperometry (CA) was of conventional design. The gas-tight electrochemical cell (volume ca. 600 pL) and the coplanar electrode assembly (edge-plane pyrolytic graphite (EPG) disk working, Ag wire quasi-reference and Pt ring auxiliary) were described previously.lq The EPG electrode areas were 0.057 f 0.002 and 0.041 (14)(a) Kessler, D. L.; Rajagopalan, K. V. J. Biol. Chem. 1972, 247, 6566-6573. (b) Johnson, J. L.; Rajagopalan, K. V.; Cohen, H. J. J . Eiol. Chem. 1974,249,5046-5055. (c) Johnson, J. L.; Rajagopalan, K. V.J. Eiol. Chem. 1977,252,2017-2025.(d) Cohen, H.J.; Fridovich, 1. J. Elol. Chem. 1971,246,359-366.
6036 The Journal of Physical Chemistry, Vol. 95, No. 15, 1991
Coury et al.
TABLE I: Metal Compkxea Used 8s Mediators for Sulfite-Reduced Chlekea L i v e r SO complex4 D, cm2/s I . [C0(3,4,7,8-Me,-phen),]~+/~+ 2.4 X IO4 2. [Co(4,7- Me,-phen),] 2+/3+ 2.4 X IOd 3. [C0(5,6-Me~-phen),]~+/~+ 3.8 X IO4 4. [Co(4- Me-phen),] W3+ 2.7 X IOd 5. [Co(5- Me-phen),] 2+/3+ 2.6 X IO4 6. [ C ~ ( p h e n ) , ] ~ + / ~ + 3.1 X IO4 7. [ R U ( N H ~ ) ~ ] ~ + / ' + 7.7 X IOdk 8. [ C 0 ( 5 - N H ~ - p h e n ) , ) ~ + ~ ~ + 4.4 X IO4 9. [ C ~ ( t e r p y ) ~ ] ~ + / ' + 4.6 X IO4 10. Fe"/"'cyt c 1.0 X 104P
E O b
-0.075 -0.052 0.014 0.039 0.096 0.152 -0.167 0.024 0.040 0.083
11. [Co(bpy),12+/'+
4.8
X
IO4
0.084
12. [ Fe(CN)6]3-/Cy 13. TMPD+/O
7.1
X
IO4
0.073
Eo'' (lit.)
5 5 5 6 16 3 5 3 6
log klZc 4.92 f 0.08 5.26 f 0.09 5.63 f 0.04 5.88 f 0.04 6.11 f 0.06 5.9 f 0.1 5.3 f 0.2 5.84 i 0.05 5.8 f 0.2 5.8 f 0.2
3 7 3
5.22 f 0.09 5.83 i 0.09 6.03 f 0.09
Nd
IO -0.037' 0.033' 0.173,'0.153' -0.127' 0.113,'-0.063" 0.0639 0.123,'-0.108" 0.219"
k2i 45h 7' 26' 45h 45h 45h 8.2 X l o z m 45h 4000 5.4 X IO3' 6 X IO2' 18' 2200" lo9"
klI8
4 (f2) x 107 5 (f2) x 108 7 ( f 2 ) x 107 4.8 (f0.9) X IO7 2.0 (fo.6) x 107 1 (*I) x 106 1.1 (f0.8) X IO8 7 ( * I ) x 107 4 (f3) x 106 8 ( f 6 ) X IO' 7 (is)X IO5 1.2 (f0.4) X IO6 see text see text
"Phen = 1,lO-phenanthroline;terpy = 2,2':6',2"-terpyridine; bpy = 2,2'-dipyridine. bThis study (0.1 M KCI, 20 mM TRIS; V vs Ag/AgCl), used k , , . CLiteraturevalues, vs Ag/AgCl (0.197 V vs NHE). dNumber of determinations of k12. 'Tolerances are 90% confidence intervals; N - 1 degrees of freedom. /Self-exchange rate constants for electron acceptors (M-' s-I). XCalculated SO apparent self-exchange rate constnat (M-I s-!); see text. hk22assumed equal to that of [ C ~ ( p h e n ) , ] ~ + /ref ~ + ;18a; (ref 3; 25 "C, 0.1 M Na2S0,). 'Reference 18b; 25 OC, pH 7.5, IO mM TRIS, 0.10 M NaNO,. )Reference 17c; pH 5.0, 5 mM NaOAc, 0.2 M NaCI. "Reference 17b; 25 OC, 0.06 M NaCI. 'Reference 21; 0.036 M NaCIO+ "Reference 22; 25 OC, 0.16 M NaOAc. "Reference 23a; 25 "C, pH 7.5.40 mM TRIS, 0.10 M NaCI. "Reference 23; 25 OC, 0.10 M ionic strength. PReference 24; 25 OC, pH 7.5, 20 mM TRIS, 0.1 M KCI. oReference 25; pH 7.0,0.5 M phosphate. 'Reference 26; 27 OC, pH 7.0,O.l M Na,PO+ SReference 3; kZ2based on cross-reaction data for [C~(phen),]'+/~+.'References 3 and 18a; 0.1 M Na2S04. "log ( k I 2 )data is UV/vis initial rate data, pH 8.45; E O ' , kZ2from ref 3; 0.1 M K2SO+ "The kZ2cited is in DMSO; its value in H 2 0 is unavailable. This kZ2varies little, however, in organic solvents with dielectric constants ranging over an order of magnitude.27 to calculate
f 0.003 cm2 as measured chronoamperometrically;'OsJ5diffusion coefficients of cobalt(I1) methylphenanthroline complexes were also measured chronoamperometrically,'S averaged over 4-5 concentrations of each complex. Redox potentials cited are the averages of E , , and E potentials in cyclic voltammograms, vs a Ag/AgCl reference erectrode (+0.197 V vs NHE)isolated from the working cell solution to avoid sulfite interference. All experiments were conducted at ambient temperature (25 "C).
Results and Discussion Electrocrtnlytic Annlysis of k I 2and k l l and Reaction Free Energy Effects. Successful electrocatalysis requires that the electrogenerated mediator be both unreactive toward sulfite substrate (in the absence of enzyme) and generated at electrode potentials at which currents for the substrate are negligible. Figure 1 illustrates these conditions. Firstly, at an edge-plane pyrolytic graphite electrode, significant current (above background, Figure 1A) for the electrochemical oxidation of sulfite (Figure 1B) starts at ca. +0.30 V vs Ag/AgCl, so mediators are restricted to those with E"' values more negative than ca. +0.2 V. Secondly, voltammetry of the metal complex [C~(phen)~]** (E"' = 0.15 V vs Ag/AgCl, Figure lC, upper curve) shows that electrogenerated [ C ~ ( p h e n )is~ ]not ~ consumed by sulfite (on the voltammetric time scale), even though [Co(phen)J3+ thermodynamically is capable of oxidizing sulfite (redox potentialI6 in basic solution = -1.13 V vs Ag/AgCl). Under the same conditions, adding enzyme as well as sulfite provokes a substantial current enhancement, Figure 1C (lower curve), reflecting the rapid electrocatalytic oxidation of sulfite via reactions 1-6. The limiting current in Figure IC (lower curve) is, at slow potential scan rates, independent of scan rate,'" signaling control by a chemical reaction rate as opposed to the rate of diffusion of sulfite to the electrode surface. Also, at sufficiently large concentrations of sulfite, the current becomes independent of sulfite (15) (a) Delahay, P . New Insrrummral Merhods in Electrochemisrry; Krieger: New York, 1980; Ibid. Interacience: New York, 1954; p 107. (b) Bard, A. J.; Faulkner, L.R. Elecrrochemical Merhods: Fundamentals and Applicarions; Wiley: New York, 1980. (c) Dclahay, P.; Stiehl, G. L. J . Am. Chem. Soc. 1952,74,3500. (d) Equation 13 of ref 15c reduces to eq 7 for 6teadyrtate cumnta and ~ ( ~ u mequal c a diffusion cccflicients for o x i d i d and reduced mediator, negligible mediatol-enzymeback-reaction, and no enzyme or rulflte concsntration polarization. All of these assumptions are reasonable and aupported by the experimental evidence. (1 6) (a) Petem, D. G.; Hayes, J. M.;Hirftje, G. M. Chemical Separations und Measunments; Ssunden: Philadelphia, PA, 1974; p AZO. (b) Klyanina, 0. L.; Shlygin, A. 1. Zh. Fiz. Khim. 1962, 36, 1310; translated in Russ. J . Phys. Chem. 1%2,36,692-694.
/
E" 0.00 6.50
.
8
bo 0
5.50
0.40
1
4
.
'0%
r:
-
0.27
0.13
7
6.00
V vs. N H E
9.10
a
'
5.00 .
O
m
7
2
0 11
1
4.50 -0.20
-0.07
E"
/
0.07
0.20
V vs. Ag/AgCI
Figure 2. Log plot of observed cross-reaction rate constants vs Eo' for 1 1 electron-transfer mediators numbered as in Table I.
concentration,'" indicating that SO is saturated with sulfite and that (6) regenerates the Enz(Fe",MoIV) form of the enzyme rapidly compared to the other reactions shown. Characteristics such as these were identified in the present study for a number of redox mediators of (2)-(6); see Table I. All yield well-defined electrocatalytic currents like that in Figure lC, including cytochrome c, the physiological electron acceptorI3for SO.Our focus is substantially on the series of cobalt phenanthrolines since their structural similarity makes them attractive for analysis of SO electron-transfer kinetics, and their diffusion coefficient^,'^ ionic electron self-exchange rate constants,17b*cJ8 and redox potential^'^-'^ are known. The appropriate relation15 for analysis of electrocatalytic steady-state limiting currents like Figure 1C is i, = nFAC*med(klZDmedC+cnz)'/2(7)
and Pen, are the concentrations (mol cm") of where Pmed mediator and enzyme,20respectively, Dd is the mediator diffusion (17) (a) Chen, Y.-W. D.; Santhanam, K. S.V.;Bard, A. J. J. Elecrrochem.
Soc. 1982,129,6146. (b) Koval, C. A.; Ketterer, M. E.; Reidsema, C. M. J . Phys. Chem. 1986, 90,4201-4205. (c) Koval, C. A.; Pravata, R. L. A.;
Reidmema, C. M. Inorg. Chem. 1984,23, 545-553. (1 8) (a) Baker, B. R.; Basolo, F.;Neumann, H. M. J . Phys. Chem. 1959, 63, 371-378. (b) Lappin, A. G.; Martone, D. P.;Osvath, P.; Marusak, R. A. Inorg. Chem. 19811,27, 1863-1868. (19) (a) Sahami, S.;Weaver, M. J. J . EieclroaMi. Chem. 1981, 122, 155-170. (b) Cummins, D.; Gray, H. B. J . Am. Chrm. Soc. 1977, 99, 5158-5167. (c) Sailasuta, N.; Anson, F. C.; Gray, H.B. J . Am. Chem. Soc. 1979, 101, 455-458. (d) Moore, G. R.; Eley, C. G. S.; Williams, G. Ado. Inorg. Bioinorg. Mech. 1984, 3, 1-89.
The Journal of Physical Chemistry, Vol. 95, No. 15, I991 6037
Electrocatalytic Oxidation of Sulfite
coefficient (cm2s-’), and k12is the bimolecular rate constant (cm3 mol-’ c’). Data for metal complexes in Table I were interpreted 7 by this relation, and as was found previously’” for [ C ~ ( b p y ) ~ ] ~ + , limiting currents are proportional to Pmd and to (CenZ)lI2.In electrochemical terms, this establi~hes’~’ that the reaction controlling the electrocatalytic current is first order each in metal complex and in enzyme, i.e., the rate constant in eq 7 is k12of reactions 2 and 4, after conversion to M-’ s-I units. 9 5 I Q 5 Thus obtained results for k are given in Table I and compared \ \ potentials. The data show that in Figure 2 to the mediator complexes with the more positive redox potentials generally exhibit larger rate constants in reactions with SO. [RU(NH~)~]~+/’+ (no. 0.00 0.15 0.30 0.00 0.15 0.30 7) has a large k I 2even though its Eo’ is not very positive, but on td30 E/V -0.65 O F ‘ AP’(;r+,yAP:, the other hand it has a larger self-exchange rate constant (k22) than do the cobalt complexes. The cross reactivities of the metal Figure 3. Catalytic behavior for [C~(Me,-phen)J*+/~+complexes numcomplex mediators with SO thus appear to depend on their pobered as in Table I. Left: Upper curve for each pair is 39 pM complex tentials, modulated by the electron self-exchange rate, as has alone; lower curve is 39 pM complex, 1 .O pM chicken liver SO and 1.4 sometimes been demonstrated for other enzymes.’ The crossmM sulfite. Center: Log rate constant (for chicken SO) vs difference in redox potentials between Co complex and Fe center in enzyme. Right: to the selfelectron-transfer rate constant kI2 is ~onnected~.’.~ Log rate constant for chicken SO plotted according to eqs 12 and 13. exchange rate constants k l l and k22 of the reactants by
?!
go’
k12 =
(8)
(kllk22Kld”2
where
K I 2= exp( E A E O ’ )
(9)
2 is the collision-limited rate constant and AEO’ the difference in reactant redox potentials. 2 = 1.8 X 1Olo M-’ s-’ based2*on a = 50.6 A for the enzyme” plus 6.4 A [Co(4,7-Me2phen),12+, Dso = 1 X lo-” m2/s,29and estimating’O zso = -2. (20) Equation 7 is derived for conditions pseudo first order in mediator. While the enzyme concentration is actually much smaller than that of the m d i t o r , the reactive Enz(Fell.MdV) form is kinetically inexhaustible because of its rapid regeneration via (6).l’l (21) (a) Brown, G. M.; Sutin, N. J . Am. Chem. Soc. 1979, IO/, 883-892. (b) . , Endicott. J. F.:Taube. H. horn. Chem. 1965. 4. 437-445. (22) Meyer, T. J.; Taube, H. InGrg. Chem. 1968.’ 7 , 2369-2379. (23) (a) Armstrong, G. D.; Sinclair-Day, J. D.; Sykes, A. G. J . Phys. Chem. 1986,90,3805-3808. (b) Drake, P. L.; Hartshorn, R. T.; McGinnis, J.; Sykcs. A. G. Inorg. Chem. 1989,28, 1361-1366. (c) Farina, R.; Wilkins, R. G. Inorg. Chem. 1968,7,514-518. (d) The k22 for [ C o ( t ~ ~ y ) ~ ] ~used +/’+ here is that reported by Baker et aI.I& after correction for ionic strength and temwrature. (24) Oliver, 8. N.; Egekeze, J. 0.; Murray, R. W. J . Am. Chem. SOC. 1988,110,2321-2322. (25) (a) Bowden, E. F.; Hawkridge, F. M.;Blount, H. N. In Comprehensive Treutise of Electrochemistrv: Srinivasan. S.. Chizmadzhev. Y, A,. Bockris. J. OM.,Conway. E. B., Ye&, E., Eds.;Plenum: New York, 1985; p 328. (b) Anderson, C. W.; Halsall, H. B.; Heinanan, W. R. Anal. Eichem. 1979, 93, 366-372. (26) Dixon, D. W.; Hong, X.; Wochler, S . E. Eiophys. J . 1989, 56, 339-35 1. (27) Grampp, G.; Jaenicke, W. J . Chem. Soc., Furuduy Truns. 2 1985,81, 1035-1 05 1 * (28) (a) 2 was estimated 2
-
K(4T)N@(D, + ~ ~ ) ( ~ ~ Z B e ’ / t k T ~ ) [ e x p ( ~ ~I]-’ ~ B ~ / f(16) k~~)
-
where K 1 for collisions between dissimilar molecules,’”bu is the sum of the radii of the two reactants (meters), D,, Dg,ZA, and z g are their diffusion coefficients (m2/s) and charges, and t is solvent dielectric constant (78.4 F m-I for H 2 0 at 25 OC).*” (b) Hammcs, 0. 0. Principles of Chemicul Kinetics; Academic: New York, 1978; pp 64-65. (c) Burgess, J. Ions in Solution: Busic Principles of Chemlcul Inteructions; Wiley: New York, 1988; pp 153-165. (d) CRC H u n d h k of Chemistry und Physics, 60th ed.; Weast, R. C., Ed.;CRC Press: Boca Raton, FL, 1980; p E-61. (29) (a) While the diffusion coefficient of SO is unknown, other wellcharacterized proteins with molecular weights ranging from 75%to 367%of that of SO (viz..108OOO for the chicken enzyme”) havem D = 1 to 2 X m2/s. (b) Van Holde, K.E. Physical Biochemistry, 2nd ed.; Prentice-Hall: Englewood Cliffs, NJ, 1985; p 103.
Line shown is weighted, linear least-squares regression for four left-hand points.
The enzyme redox potential is taken as that of its cytochrome bs-like center,” -0.1 13 V vs Ag/AgCl (+O.O84V vs NHE), which as shown later is the cross-reaction site. Equations 8-10, using the literature k22values for the metal complexes and the experimental kI2 and Mor(Table I), produce the apparentg2enzyme electron self-exchange rate constants, kll, shown at the right of Table I. For the cobalt complexes 1-5,7, and 8, the apparent kll values are reasonably constant with a mean of 1.2 X lo8 M-’ s-’ (reciprocal variance-weighted mean = 3.9 X lo7 M-l s-’). This rate constant is that which would be exhibited for electron exchange between two sulfite oxidase molecules were their heme sites (hypothetically) to have, both sterically and electrostatically,the same “accessibility” to one another as do these metal complexes to the heme site.’ We will return to these problems in a later section. The series of cobalt complexes 1-6 involves minimal structural and no charge variations and allows a closer examination of the reaction free energy dependence of klz. Figure 3 (left and center) illustrates the systematic increase of electrocatalytic currents for Eo’ becomes more positive. Their these mediators as the CO”’/~~ behavior is plotted in Figure 3 (right) using a rearranged form37 of ws 8-10 log k12 = 0.5 log (kIIk22) 8.45AE0’[1 xAE0’] (11) where
+
+
(12)
In the plot shown, kllk22(and hence x) are iterated for the best fit to the log ( k I 2 )points for complexes 1-4. The least-squares (30) (a) Speck, S. H.; Koppcnol, W. H.; Dethmers, J. K.; Osheroff, N.; Margoliash, E.; Rajagopalan, K. V. J . Eiol. Chem. 1981,256,7394-7400. (b) There is some evidence that the SO binding site for cytochromec has a charge as low as -2. However, if zso is assumed to be as large as -13 (the charge based on the ionizable amino acid rtsiduesMsin the heme domain of SO), the calculated z increases by only 3-fold, which introduces a negligible change in f i n eq IO. (c) Guiard, B.; Lederer, F. Eur. J . Biochem. 1977,74, 181-190. (d) For the cyt c crws reaction, 2 = 1.03 X IOio M-l S-I assuming a charge and radius for cyt c of +6.5 and 16.6 A, (31) Cramer, S.P.;Gray, H.6.;Scott, N. S.;Barber, M.;Rajagopalan, K. V. In Molybdenum Chemistry of Eiologfccrl Sigmpcunce;Newton, W. E., Otsuka, S.,Eds.; Plenum: New York, 1979; pp 157-168. (32) We refer to kll as appurent not only because accessibility and electrostatic terms remain uncorrected, but also because the poasible role of precursor complex formation prior to electron transfer’ is neglected. If this occurs, k I 2= K k,,, where Kpo is the equilibrium constant for precursor formation. WhEvariations in Kpo could in principle introduce scatter into the calculated enzyme k l l values, it should be constant within the series of methyl-substituted phenanthroline cobalt complexes given their chemical and physical similarities. (33) Leidner, C. R.; Murray, R. W. J . Am. Chem. Soc. 1984, 106, 1606-1 61 4.
6038 The Journal of Physical Chemistry, Vol. 95, No. 15, 1991 slope (8.78) for these complexes is close to the theoretical one, 8.45,and the intercept, 4.64,corresponds to an apparent enzyme k l l of 4 X IO7 M-' s-I, taking3JBak22= 45 M-' s-I. This k l l is indistinguishable from the weighted mean value derived above. The successful Marcus-Hush2 analysis of Figure 3 (right), of the free energy dependence of enzyme/cobalt complex electrontransfer cross-reaction rates, argues that kI2directly reflects the rate of electron delivery from heme to cobalt complex as opposed to some other rate step such as adduct formation. The analysis deviates, however, for differences in Eo' values greater than ca. 0.2V. The rate constants observed for cobalt complexes with more positive Eo' seem to level off at an apparent maximum value. Unless the reorganizational barrier for this reaction is exceptionally small (Le., near 0.25eV) in comparison to other known heme protein reactions, the leveling-off of kI2is difficult to understand on a reaction free energy basis. In this connection, it is revealing to note (Table I) that kI2appears to attain a similar upper limit, ca. 1 X IO6 M-' s-', under three other circumstances where the heme site turnover is forced toward larger absolute rates. These are (i) cross reactions with a mediator of even more positive Eo' ([Fe(CN)6]s/-/t; in this case a spectrophotometricallyderived k , J , (ii) with an organic acceptor of modest Eo' but very large27kz2 (viz., [TMPD]+/O),and (iii) at low ionic strengths (vide infra) that electrostatically enhance the reaction rate. Since the electrocatalytic chemistry involves multiple reaction steps, an obvious possibility is that kinetic control of catalytic currents shifts to another of the reaction steps when the rate for reaction 2 exceeds ca. 1 X IO6 M-' s-'. Let us examine the intramolecular electron-transfer steps, reactions 3 and 5, with this possibility in mind. Internal electron-transfer rates have been studied for sulfite oxidase following reaction with photolytically generated flavin radicals." Rate constants for internal Mo Fe and Fe Mo electron transfers were reported9' as kf = 3 IO s-' and kb = 155 s-I, respectively. For comparison to the electrochemical situation, given that the enzyme should initially be in the fully reduced form (Enz(FeI1,MdV)), in the presence of excess sulfite, a 0.8 pM formal enzyme concentration corresponds to 0.8 pM enzyme Mo(IV) and Fe( 11) concentrations. Upon electrogeneration of oxidized mediator, the internal Mo Fe electron flow can occur at a velocity of u = kf[Mod] = (310 s-')(8 X lV7M) = 24 X lVs M s-l. For comparison, in the case of a second-order reaction with rate constant k I 2= 1.3 X lo6 M-' s-' (e.g., [Co(5-Me-phen),13+reacting with the enzyme) at a 39 pM mediator concentration, generation of Fe(II1) states occurs with a reaction velocity u = k12[med][enz]= (1.3 X 106 M-' s-')(3.9 X lV5 M)(8 X lV7M) =4X M d. That is, when a fast mediator is used in reactions 2 and 4,the turnover of heme sites is forced to within a factor of 6 of the velocity with which the reduced heme sites can be regenerated by internal redox reactions 3 and 5 according to literature data" for the rate of those steps. At higher mediator concentrations,or with even more potent mediator-xidants" (Le., [Fe(CN)6]% and TMPD"), the heme turnover rate would even more closely approach the internal forward electron-transfer velocity. This comparison indicates that the deviation from Marcus-Hush behavior in Figure 3 (right) arises from encountering a shift of rate control to another reaction step and suggests (but does not prove) control by the internal electron tran~fer.,~ Significance of k 12 and k I I : Accessibility and Ionic Strength Effects. As noted above, k l l = 4 X IO7 M-' s-I corresponds to
-
-
-
(34) k,,values for SO reacting with mediators 12 and 13 a n not estimated in Table 1 for this reason. (35) A crude estimate of the Mo-Fe distance (d) in the enzyme may be obtained from'
b estimating the rc4r nization energy, A to be equal to that for free cytoclrome bJ (I ,158 x itY~,m013); = 0.9 A-I as as sum at^*^ for cytochromes c and -bp and calculating AGO from the difference in redox potentials of the MoV/lvand Felll/llsites (Mol= 0.079 V)." Basedh on kr = 310 s-l, d is CB. 13 A. The detailed structure of SO is not known, but given that it is a dimer with a Stokes radius of 50.6 A," this d seems reasonable.
Coury et al.
I
pA/cm'
m toa
-0 I
E /
\
\s
hg/AgCI
Figure 4. Effect of ionic strength on electrocatalytic voltammetry, 2 mV/s using (A) [ C o ( d p ~ ) , ] ~ - /(B) ~ , [Co(bpy),12+/'+, and (C) [Co(phen),12+13+as mediators. In each case, [Co] = 72 cM, [enz] = 3 cM, and [SO3*-]= 1.4 mM; for (B) and (C), upper curve is CV of mediator alone. Ionic strengths from left to right, (A): p = 0.10,0.17,0.27,0.43, 0.55, 3.0, 4.0 M. (B) and (C): 1 = 0.10, 0.17, 0.27, 0.43, 0.55 M.
the rate constant of a hypothetical electron self-exchange between two sulfite oxidase molecules having the same mutual steric accessibility as well as the same electrostatic interactions as do the metal complexes with the heme site., Likewise, kI2for the cobalt complex/enzyme reaction is uncorrected for any steric modulation of accessibility to the heme reaction site or for electrostatic interactions with it. We consider these two effects next. Accessibility is known to be an important i ~ s u ein ~ .enzyme ~ electron transfers. In the present case, we see that the metal complex-derived k l l for SO is larger than the k l l derived from cytochrome c kinetic data (Table I, see no. 10). Accessibility encompasses, among other factors? (i) steric effects associated with the fraction of the protein surface active toward electron transfer in a collision encounter,' and (ii) the distance dependence of electron The steric factor is known' to have a considerable effect in direct protein self-exchange. Unfortunately, the accessibility problem cannot be directly evaluated for sulfite oxidase; its crystallographic structure is not available. We can say that the numerical value of the apparent SO kll is similar to k l l results for non-proteinaceous porphyrins analogous to the SO heme (FelIll" protoporphyrin-IX-bi(histidy1)) site.,& For example,36k l l = 8 X IO7 M-' s-I for [FeI1ln1tetraphenylporphyrin-bis(l-Me-imidazole)l0/+ and exceeds 1 X lo7 M-' s-l for [ FelI/II1 tetrakis(N-methylpyridinium-4-y1)porphyrin-bis(imida~ole)]'+/~+. It could be inferred from these kinetic similaritiesthat the kll measured here for SO heme reflects relatively free access of the cobalt complexes to the heme site. This picture is severely clouded, however, by the substantial effect that ionic strength has on measured kI2values, as reported next. Coulombic interactions between cobalt complexes and reduced sulfite oxidase were investigated in 20 mM TRIS, using varying ionic strengths of KCI. When a negatively charged complex, ( [ C o ( d p ~ ) ~ dps ] ~ /=~4,7-diphenylphenanthrolinedisulfonic , acid) is used, the electrocatalytic currents (Figure 4A) clearly were increased at larger ionic strengths, but unfortunately were too poorly defined to yield k12data. The cationic complexes ([Co(bpy),]*+/'+ and [Co(phen),]*+/)+),on the other hand, produce well-defined, steady-state catalytic currents (Figure 4B,C)that steadily decrease as p increases from 0.1 to 0.5 M. These ob(36) (a) At 0.12 M ionic strength, k l l for cyt b, is" 3 X lo3 M-i &, and upon extrapolation to infinite ionic strength, k l l values for cyt bJ, -c, and -cJll are reported" as 4 X IO', 5 X IO', and 2 X 10' M-' s-I, rmpcctively. (b) Feng, D.; Schultr, F. A. Inorg. Chcm. 1988, 27, 2144-2149.
The Journal of Physical Chemistry, Vol. 95, No. 15, 1991 6039
Electrocatalytic Oxidation of Sulfite
TABLE 11: Eketroatatie Work Tenns, ColIlsioa Factors, lad Corrected SO ScH-Excb.ngc Rate Comtrab ZW'
Rw'
-2
5 IO 5 IO 5
-5 -6
&Ib
1.43 X IO9
ZIP 1.78 X 1O'O
7.02X IO'
2.77 X
1Olo
4.49 X IO'
3.15 X
1Olo
1.22 X IO'
3.95 X 10"
10
-8
5
IO
W1zC
-0,7006
-0.3298 -1.7515 -0.8244 -2.1018 -0.9893 -2.8024 -1.3191
%IC
-0.2335 -0.1099 -0.9341 -0.4397 -1.1677 -0.5496 -1.6348 -0.7695
AGlzbC 6.858
WllC
0.3313 0.0734 3.3129 0.7338 4.9693 1.1006 9.2761 2.0545
7.121 7.196 7.331
AGli*'
hOmf
9.539 8.786 14.80 10.80 17.19 11.59 22.93 13.36
1.44 X 5.13 X 9.83 X 8.46 1.11 x 1.42 1.84 X 1.92 X
102 IO2 IW3 10-4
IO* 1W2
'Assumed values for anion patch charge and radius (A) of heme reaction site. bCollision factors (M-I s-l) calculated by eq 16 using enzyme radius of 50.6 A. Z2; = 8.94 X 10' M-' cI. CWorkterms (kcal/mol) calculated by eq 14 for p = 0.1 M. wz2 = 0.507kcal/mol. dActivation free energy for cross reaction (kcal/mol). AGZ2* = 10.49 kcal/mol. AGi20 = -nFAE0' = -4.543 kcal/mol. 'SO self-exchange activation energy (kcal/mol) calculated from eq 15. fcorrected SO self-exchange rate constant (M-I s-I). servations strongly suggest that the site of electron transfer between enzyme and metal complex (the heme site: vide infra) is anionic. That is, the higher ionic strengths in Figure 4 provide an ionic atmosphere that screens attractive electrostatic interactions between anionic enzyme active site and cationic metal complex mediator and slows the rate. A quantitative interpretation of the ionic strength effect in Figure 4B,C require^^,^.^ an estimation of the anionic charge at the enzyme heme site and a guess at the size of the anionic patch (R,). While the detailed structure of sulfite oxidase is unknown, the amino acid residues which comprise the heme domain of the enzyme have been determined,* and at the pH employed, comprise a total charge of -13. From the crystal structure3' for Fe(prot0porphyrin-IX)bis( 1-methylimidazole),the small molecule analogue of the SO heme, the Fe porphyrin is 3-4 A wide (excluding side chains), which sets R,, = 4 A and z,, = -13 as the lower and upper limits of heme site size and anionic patch charge, respectively. The problem was further investigated by assuming various values for Re,, in the ionic strength function,flp), of Wherland and
14
13
Ink,,
l2 11
'
10 -0.6
-0.2
-0.4
00
ionic s t r . f u n c t i o n Figure 5. Log plot of klz measured for [C~(bpy),]~+ from Figure 4B vs ionic strength function defined in eq 13. Inset: Plot of squared cone-
lation coefficients for regression of In (kI2) ontoflp) for different values of effective enzyme radius L.
C o ( p h e n ) 33t
I
j@) = -3.576
(13) where K = 0.329(~)~/~ A-l. Figure 5 shows a linear, least-squares fit of k I 2constants derived from Figure 4B for [ C ~ ( b p y ) ~ ] ~ + / ' + againstflp), assuming Re,, = 5 A. The slope of this plot yields z , = -6.0,and itsfi) = 0 intercept gives an infinite ionic strength rate constant (i.e., all electrostaticcontributions are screened out) kI2 = k, = 1.5 X IO' M-'s-l, The Figure 5 inset shows the squared correlation coefficients (R2) for a series of eq 13 plots that assumed the indicated values for R,,. While the maximum = 5 A, which is statistically the best value, R2 is obtained for R2 actually varies little between R,, = 3 and ca. 9 A. Assuming R, = 6-9 A gives z , = -6.8,-7.7,-8.7,and -9.8,respectively. We are left with considerable uncertainty in these values, but it does appear that the heme reaction site bears a substantial negative charge. These estimates for Re, and z , may be em loyed to calculate a k l l unenhanced by electrostatic a t t r a ~ t i o n s J ' Using ~ ~ the expression
1n k;, 14 IC,
l3 12
\
r
-0.4
-0.3
-0.2
-0 1
00
ionic s t r . function Figure 6. Log plot of k12measured for [C~(phen)~]~+ from Figure 4C vs ionic strength function defined in eq 13. Line is regression calculated for rightmost three points.
where AG,]* = RT(ln Z , - In k,,) and AGlz0 = -nFAEo'. Combining these values results in the corrected AGll* and kllm values in Table 11. It is immediately obvious that the electrostatic effect on k l l is both very large and highly sensitive to the choices of anionic patch charge and size. Additionally, in all cases, even assuming a small zm,the kllm values are much less than monomer porphyrinic kll rate constant~,'~ from which one could speculate that the SO heme reaction site is actually sterically not very accessible and that the fast SO reaction kinetics are s u b stantially due to its favorable electrostatic interactions. Further w,,, the work terms describing electrostatic interaction between understanding of this problem must await structural information species i and j at 25 OC in aqueous medium, were calculated (Table for the enzyme. 11) for two different assumed enzyme radii and selected enzyme do not produce linear plots reaction site charges, and employed to calculate a c o r r ~ c t e d ~ , ~ * ~ The kI2results for [Co(ph~n)~]~+/'+ (Figure 6)of eq 13 for any physically meaningful assumptions SO self-exchange activation energy (AGII*)by of R, (is., R, > 3 A). It is notable that kI2deviates from the ~ 2 2 AGII* 2AG12* - AG22* - AGizO- ~ 2 -1 ~ 1 +2 w I I electrostatic theory at the lowest p (more negativeflp)) and also (15) that [ C ~ ( p h e n ) J ~ + is / ~the + most oxidizing (most positive E O ' ) of the metal complexes (Table I), exhibiting kinetic saturation and plateauing at high reaction velocities as discussed above. (37) Little. R.G.; Dymock, K.R.;Ibers, J. A. J . Am. Chcm. Soc. 1975, 97,4532-4539. These two observations are entirely self-consistent: encounter of
+
a40
J . Phys. Chem. 1991, 95, 6040-6044
rate limitation by internal electron transfer would interrupt an expected increase in klzat low ionic strength, provided the internal reaction is less affected by variations in ionic strength. Elucidation of Site of Reactivity with Electron Acceptors. The preceding analysis demands evidence that the measured kI2values correspond to reaction exclusively at the SO heme site on our experimental time scale. Sulfite oxidase is a dimer of two identical subunits, each of which contains a Mo atom present as a molybdopterin c ~ f a c t o r , ' ~the , ~ site ~ at which oxidation of sulfite occurs.13 Each subunit also contains the above mentioned cyt bs-like center where reduction of the physiological electron acceptor, cytochrome c, occurs subsequent to an internal charge transfer.I3 Cleavage of the holoenzyme into Mo- and Fe-containing domains by trypsin and isolation of the Mo fragment yields a sulfite-reducible molybdoprotein which has lost the ability to reduce cytochrome c, but which retains a level of ferricyanide reductase activity which is virtually unchanged compared to that observed for the h ~ l o e n z y m e . ~ ~ ~ ~ " Against this established background, we tested whether various metal complex electron acceptors catalytically oxidize sulfite in the presence of the molybdoprotein fragment, using voltammetric experiments conducted as described above. No catalytic currents are observed in mixtures of Mo fragment, sulfite, and mediator, for [C0(4,FMe~-phen)~] 2+/3+, [Co(bpyI3]2+/3+, [Ru(NH3),] '+I3+, or the anionic complex [ C o ( d p ~ ) ~ ] & (vide / ~ supra). The ability
of the sulfite-reduced fragment to reduce ferricyanide was confirmed both electrochemically and spectrophotometrically, thus demonstrating that the Mo fragment sample was active. From this we infer that the reaction site of these mediators (except [Fe(CN)6]*'/3-) is the enzymic Fe(I1) site, as is the case for the physiological acceptor, cytochrome c.13 It should also be noted that no evidence was found for direct electron transfer between the EPG electrode and the Mo fragment, either in the presence or in the absence of sulfite. Acknowledgment. This research was supported in part by grants from the National Science Foundation and the North Carolina Biotechnology Center. Registry No. [C0(3,4,7,8-Me-phen),]~+, 47889-06-5; [Co(3,4,7,8Me,-phen),]'+, 86176-94-5; [C0(4,7-Me,-phen),]~+,47872-45-7; [Co(4,7-MeZ-phen),]'+,62791-75-7; [Co(5,6-Mez-phen),l2+,47872-55-9; [Co(5,6-Mez-phen)#+, 62869-82-3; [Co(4-Me-phen),lZ+,8071 1- 1 I- 1; [Co(4-Me-phen),13+,807 1 I - 14-4; [Co(5-Me-phen),lZ+,47860-25-3; [Co(S-Me-phen),lZ+,96504-30-2; [Co(phen)J2+, 16788-34-4; [Co(phen),])+. 18581-79-8; [ R u ( N H ~ ) ~ ]19052-44-9; ~+, [RU(NH,)~]~+, 18943-33-4; [C0(5-NH~-phen)~]~+, 1 13442-71-0;[Co(S-NHZ-phen),l3+, 1 13461-17-9; [C~(terpy)~]*+, 18308-16-2; [C~(terpy)~]'+, 19137-07-6; F ~ y t c9007-43-6; , [Co(bpy)J", 15878-95-2;[C0(bpy),l3+, 19052-39-2; [ Fe(CN)613-, 13408-62-3; [ Fe(CN),]@, 13408-63-4; TMPD", 3452755-4; TMPLY', 100-22-1;SO,9029-38-3; molybdoheme, 126858-98-8; sulfite. 14265-45-3.
Determination of Water Dlffusion Coefficients in Perfluorosulfonate Ionomeric Membranes Thomas A. Zawodzinski, Jr.,* Michal Neeman, Laurel 0. Sillerud, and Shimshon Cottesfeld Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (Received: September 17, 1990; In Final Form: March 18, 1991)
Pulsed field gradient spin-echo 'HNMR measurementsof 'Hintradiffusioncoefficients at 30 "C in hydrated Nafion membranes are reported. The dependence of the 'Hself-diffusion coefficient on membrane water content was a central part of this investigation. 'Hdiffusion coefficientsranged from 0.6 X 10-6 to 5.8 X IOd cmz/s for the range of membrane water content 2-14 water molecules per sulfonate. The membrane water content was controlled by isopiestic equilibration of the membrane sample with water vapor above aqueous LiCl solutions of well-defined water activities. The dependence of membrane water content on water activity enables us to estimate "chemical diffusion coefficients" from the intradiffusion coefficients measured by NMR.
Introduction Fuel cells using perfluorosulfonate ionomers (e.g. Nafion) as proton (H+) conducting electrolytes are attractive candidates for use in electric vehicles.' In our laboratory, such fuel cells are constructed by hot-pressing together a "sandwich" consisting of an ionomeric membrane separating a pair of gas diffusion electrodes impregnated with the recast ionomer.2 The only liquid added to this system in order to operate it as a H2/02 fuel cell is distilled water. Water is supplied to the fuel cell by humidifying the gas feed stream. Adequate water content of the ionomeric material is essential to maintain the conductivity of the polymer electrolyte membrane. This has, in turn, a strong effect on cell performance. However, excessive amounts of liquid water could impede mass transport within the electrode structure. As part of a larger effort directed at understanding the role of water ( I ) Lemons, R. J. Power Sources 1990, 29, 251. (2) (a) Raistrick, 1. D. In Diaphragms, Separators, and Ion-Exchange Membranes; Van Zee,J. W., White, R. E., Kinoshita, K., Burney, H.S.,Eds.; The Electrochemical Society: Pennington, NJ, 1985; p 172. (b) Ticianelli, E. A.;Derouin, C.R.; Srinivasan, S.J . Electrmnanl. Chem. 1988, 251, 275. (c) Gottesfeld.S.; Raistrick, 1. D.; Srinivasan, S.;Springer, T. E.; Ticianelli. E.; Detouin, C. R.;eeCry,J. 0.;Pafford, J.; Sherman, R. J. Presented at the 1988 Fuel Cell Seminar, Long Beach, CA.
0022-3654/9 1/2095-6040$02.50/0
content and possible modes of water management in polymer electrolyte fuel cells, we are engaged in a variety of experimental studies of the properties of water in perfluorosulfonate ionomers with the goal of developing models of water distribution across a fuel cell under typical operating conditions. The diffusion coefficient of water in Nafion and related membranes as a function of water content is a necessary input for the analysis of the performance of cells based on these membranes. Nafion-based polymer electrolyte fuel cells (PEFC) are usually operated under conditions of partial hydration of the polymer. Water is lost from the membrane during fabrication of the membrane/electr& assembly and is apparently not fully replaced by humidification of the membrane with water vapor carried into the fuel cell with the reactant gases. The level of hydration could also vary significantly with position in the membrane when the cell is under current. A nonuniform water concentration profile is set up in the membrane by an electroosmotic drag of water from anode to cathode and by the production of water a t the cathode. The water concentration gradient set by these two effects is expected to be made less steep due to water "back-diffusion" from cathode to anode. Clarification of the water profile across the ionomeric membrane in a cell under current thus requires knowledge of water diffusion coefficients as a function of water 0 1991 American Chemical Society
