J. Phys. Chem. 1993,97, 1694-1700
1694
Intra-Enzyme and Mediator Cross-Reaction Electron-Transfer Reaction Kinetics of Sulfite Oxidase Liu Yang, Louis A. Coury, Jr.,t and Royce W . Mumy’ Kenan Laboratories of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290 Received: September 17, 1992; In Final Form: November 12, 1992
At saturating concentrations of sulfite, the consumption of electron-transfer mediator-xidants by reaction with the heme site of sulfite oxidase is governed, a t low turnover rates, by the mediator-heme electron-transfer cross-reaction rate and, a t high turnover rates, by that of the intra-enzyme delivery of electrons from enzyme Mo sites to heme sites. The mediators employed are cobalt polypyridine complexes and ferricytochrome c. Rate constants (k12)for mediator-heme cross-reactions are shown to depend on the choice of mediator, whereas rate constants (kin) for the intra-enzyme reaction do not. Spectrophotometricmeasurements confirm the mediated electrocatalysis results in the case of the mediator-oxidant [Co(terpy)213+. Values of k12 among a series of cobalt phenanthroline electron acceptors vary with the reaction free energy quantitatively as predicted by Marcus theory, The effects of pH, added chloride, and ionic strength on the rate constants have also been
examined. Sulfite oxidase is a molybdohemoprotein that catalyzes the oxidation of sulfite to sulfate.’ The enzyme is a dimer of two identical subunits, each of which containsa molybdenum cofactor2 and a cytochromeb5-type heme.3 The two-electron reduction of the Mo(V1) site by sulfite is followed by two sequential intraenzyme electron transfers from the Mo(1V) and Mo(V) states to the heme site, which is in turn oxidized by cytochrome c (in vivo) or by synthetic mediator-oxidants. There have been studies of both electron-transfer steps. That for the intra-enzymeelectron transfer has been examined4using photolytically generated flavin radicals; the kin’s for Mo Fe and Fe Mo electron transfers were reported as kf = 310 s-I and kb = 155 s-1, respectively. We have e m p l ~ y e dmediated ~.~ electrocatalysis to investigate electron-transfer cross-reactions between the enzyme heme site and a variety of mediator-oxidant species. In a series of [Co(Me,phen)#+ mediators, for the slowly reacting members of the series, the mediator-nzyme electrontransfer cross-reactionis the rate-determining step and the rate constants k12follow classical reaction rate-free-energy theorye6 For faster reacting mediators, the apparent cross-reaction rate constants, on the other hand, appeared to approach limiting values, suggesting a shift of reaction rate control to another reaction step, possibly that of the intra-enzymeMo+ Fe electrontransfer. The present study explores this change of reaction rate control. We describe here mediated electrocatalytic and spectrophotometric results for the rates of cross-reactionsof the sulfiteoxidase heme site, under sulfite-saturated conditions, with the mediatoroxidants [Co(terpy)2]3+, [Co(bpy)#+, ferricytochrome c, and the previously employed6series of [Co(Me,phen),] 3+ complexes. The plateauing of the reaction rate observed at high enzyme turnover rate is analyzed as two serial electron-transfer reactions: one with a rate constant that limits reaction velocity under fast reaction conditions and which proves to be independent of the mediator employed and another with a bimolecular rate constant (kl2) for the mediator-heme site cross-reaction which isobserved tovary with themediator employed. The fast reactionlimiting, mediator-independent rate step (kin)is proposed to be that of the intra-enzyme Mo Fe electron transfer. The improved values for k12 of the mediator-xidants previously investigated6,including those for the series of [Co(Me,phen)3] 3+ mediators, are shown to follow classical reaction rate-free-energy theory for both slow- and fast-reacting mediators.
-
+
-
’ Present address: Department of Chemistry,Duke University,NC 27706. 0022-3654/93/2097- 1694$04.00/0
The effects of buffer composition, added chloride, and pH on the intra-enzymerate constantand heme-mediator cross-reaction rate constants are also reported. The kinetic effects seen are ascribed primarily to variations in the formal potentials of the Mo site in the enzyme. When [Co(terpy)2l3+is used as the mediator, the product, [C0(terpy)~]2+,is a mild inhibitor for the enzyme reaction, possibly through enhancingthe rate of the backreaction direction of the electron-transfer cross-reaction.
Experimental Section Sulfite oxidase was isolated from chicken liver as previously described7and stored in 1-mL quantities at 0 OC. The ferricyanide reductase activity of three different batches of the enzyme was spectrophotometricallyassayed7in pH 8.5,O. 1 M TRIS/100 pM EDTA (TRIS = tris(hydroxymethyl)aminomethane), using initial rate measurements and saturating concentrationsof sulfite. The second-order rate constant for the reduction of ferricyanide by reduced sulfite oxidase was (1.0 f 0.1) X lo6 M-l s-l for batch A of the enzyme, (4.9 f 0.2) X lo5M-’ s-1 for batch B, and (1.07 f 0.02) X 106 M-I s-1 for batch C. Cytochrome c (Type VI, horse heart, prepared without trichloroacetic acid) was obtained from Sigma and used without further purification. Solutions of Co(I1) complexeswere prepared in situ from CoClr6H20 and a small excess of ligand in N2flushed vials. Ligands (G. F. Smith Chemicals, Aldrich, Alfa, PolySciences) wereused as received. [Zn(terpy)2]2+was prepared by mixing ZnCI2 and the ligand (terpy = 2,2’,2”-terpyridine) in 0.02 M TRIS/O.l M KCI buffer, pH 8.5, followed by warming at 60 OC for 10 min. These solutions were employed in the mediated electrocatalysis measurements. [C0(terpy)~]3+solutions, used in spectrophotometric rate measurements, were prepared by electrochemicaloxidation of [Co(terpy)2I2+at +0.500 V vs Ag/AgCl. All other chemicals were high purity, commercially available. Unless otherwise noted, thoroughly degassed solutions for electrochemistryand spectrophotometrycontained 0.02 M TRIS/O.l M KC1 or 20 mM KH2P04. Adjustment of the pH was accomplished with added HCl or KOH for TRIS/ KCI buffer and with KOH for phosphate buffer. The electrochemicalcellS (volume ca. 600 rL) was gas-tight with coplanar edge-plane pyrolytic graphite working electrode (EPG, chronoamperometricallymeasured area of (2.9 f 0.2) X 10-2 cm2) and Pt ring auxiliary electrode and with a Ag/AgCl reference electrode (0.1 M KCI) compartment isolated from the protein-containing solution. Instrumentation for cyclic Q 1993 American Chemical Society
The Journal of Physical Chemistry, Vol. 97, No. 8,I993
Reaction Kinetics of Sulfite Oxidase voltammetry was of conventional design. Temperature was maintained at 25 OC. Values of steady-state electrocatalytic currents for [Co(terpy)#+, [Co(bpy)3]2+,and ferricytochrome c were obtained in slow-potential scan cyclic voltammetry. Those for the [Co(Me,~hen)~] 2+ complexeswere obtained following potential steps from -0.1 V (mediator in reduced state) to +0.2 V vs Ag/AgCl (suffcient tooxidize the mediator but not sulfite6);steady currents are observed shortly after the potential step. Experiments with [Co(terpy)~]~+ using these two procedures verify that they give the same result. Calculations of theoretical steady-state electrocatalytic currents were done by digital simulation of diffusion equations written to represent a series of two-electron-transfer steps. The diffusion concentration profile was calculated first; the concentration arrays were then modified by the enzyme reaction.* The experimental rate constants were estimated by comparing simulated catalytic currents to the dependence of experimental catalytic currents on mediator reactant concentrations. Spectrophotometric reaction rate measurements using [Co( t e r p ~ ) ~as] ~the + mediator-oxidant were conductedanaerobically at ambient temperature with an HP-8452A photodiode-array spectrometer interfaced to a laboratory PC. The initial enzyme reaction rate was determined by the decrease in absorbance at 352 nm, from the reduction of [Co(terpy)2I3+ that followed addition of Na2S03to the solution of buffer, sulfite oxidase and [Co(terpy)2]3+. The molar absorbance coefficient of [Co(terpy)213+at 352 nm is 3970 M-' cm-'.
Results and Discussion Elucidation of Rate Law. The well-established catalytic cycle for sulfite oxidase reactionsi, k12
( I ~v )R E+DE ~ z F ~ ( I I I ) , M(1) ~(Iv) M d o x + E ~ z F ~ ( I I ) , M ~M -+
km
E ~ z F ~ ( I I I ) , M E~ ~( IzvF)~ ( I I ) , M ~ ( v )
(2)
k12
M d o x + E ~ z F ~ ( I I ) ,+ M ~M( v~) ~ R+EED~ z F ~ ( I I I ) , M ~ (3) (v) kin
E ~ z F ~ ( I I I ) , M ~E(~vz) F ~ ( I I ) , M ~ ( v I ) +
En+e(i1),Mo(vi)
+ SO,'- + H2O
-.
EnZFc(ll),Mo(lV)
+
(4)
+ 2H+ (5)
is abbreviated in our analysis as
M d o x + EnzRED
k12 +
MedRED + Enzox
(6)
where Medox represents the oxidized form of the mediator, Le., [C ~ ( t e r p y )3+, ~ ] [Co(bpy)3]3+, [Co(Me,phen) l] l+, or ferricytochrome c. EnzRED and EnQx represent the heme-reduced and heme-oxidized forms, respectively, of the enzyme. No attempt is made in the internal Mo Fe electron-transfer step (kin) to distinguish between transfers to heme from the Mo(1V) vs from the Mo(V) state, and k12is assumed to be the same in reaction 1 and 3. We have established6 the absence of reaction (on the voltammetric time scale) between cobalt complex mediator and theenzymehfosite. The presenceof a saturatingexcessof sulfite means that reaction 5 is very fast and EnzFe(ll),Mo(VI) is quickly turned over to the Mo-reduced EnzFc(Il),Mo(lV)state. Thus, following oxidation of the heme site of the enzyme (EnzRED) by the mediator in reaction 6, regeneration of EnZKED is governed by the intra-enzyme Mo Fe reaction, (7). It can be anticipated
-
-
16%
I
I
$0.2s
'0
EN
-0.B
Figure 1. Voltammogramsat 2 mV/s for 0.43 mM [Co(terpy)~]*+ alone (uppet curve) and in the presence of 1.O pM (batch A) enzyme and 13.8 mM sulfite (lower curve). Cross represents 0 V vs Ag/AgCI and no net current.
that the overall rate of oxidized mediator (Medox) consumption will be governed by reaction 6 when the mediator concentration [Medox] or rate constant k12is small and by reaction 7 when they are large. The initial velocity of the enzyme reaction for reaction 6
= k,2[Medoxl [EnzREDl
(8) and the steady-state condition for EnQx once the reaction begins
d[EnzOXl/dz = k12[MedoXl [EnzRE,l - k~n[EnzOXl= 0 ( 9 ) can be combined to give an expression for the reaction velocity which in reciprocal form is
-=-+1
1 1 1 (10) Vel kinCcnz* k~tCenz*[MdOxI where Cenz*is the total enzyme concentration. This relation predicts a linear reciprocal relation between reaction velocity and mediator concentration. Rate Measurements by Mediated Elecbocatdyris. Mediators suitable for electrochemical detection of the rates of reactions 6 and 7 must meet the criteriaS of (i) well-defined, chemically reversible voltammetry, (ii) negligible rate (on the voltammetric time scale) of direct reactivity with the enzymatic substrate (viz., sulfite), and (iii) rapid electron-transfer cross-reaction rate with the reduced enzyme. We have shown5s6that cobalt polypyridine complexes meet these criteria. [ C ~ ( t e r p y ) ~was ] ~ +attractive for a detailed kinetic study, being one of the faster reacting complexes6 and having an optical absorbance suitable for spectrophotometric kinetic measurements for comparison to the electrocatalytic results. The electrochemical experiment is based on the voltammetric current for oxidation of mediator (e.g., [C0(terpy)~]2+).Voltammetry of this mediator in the absence of enzyme is shown in Figure 1, upper, whereas Figure 1, lower, shows the enhanced current caused by the regeneration of [Co(terpy)#+ by reactions 6 and 7 in the diffusion layer around the electrode when sulfite oxidase and a limiting excess of sulfite are also present in the solution. On a sufficiently slow potential sweep time scale, the electrocatalytic current has a steady-state value. Voltammetric theory for a mediated electrocatalytic reaction involving a single rate-controlling step, such as the cross-reaction reaction 6, predicts*that the limiting current for a voltammogram like Figure 1, lower, should vary proportionately with mediator concentration according to the relation i,, = nFACmd* (ki2DmdCenz*)1/2
(1 1)
16%
-a Y
Yang et al.
The Journal of Physical Chemistry, Vo1. 97, No. 8, 1993 o.80
8E. m
L.
0.75
' m
C
0
cI
0 0 0
= 0.0et00
2.3e-05
4.5e-05
6.78-05
9.0e-05
0.25
0.00
'
1
0.0.+00
2.01-05
electrocatalytic plateau currents. ( 0 )Experimental current at 2 mV/s, with 0.68 pM batch B enzyme, 6.3 mM sulfite; (+) calculated currents based on k12 = 1.2 X IO7 M-I s-l and kin = 131 s-1.
z
h
(12) This quation accountsfor the electrochemicaldiffusion problem and the two enzyme reaction steps of reactions 6 and 7. Digital simulation calculations(see ExperimentalSection) of the steadystate electrocatalytic limiting current for chosen values of the rate constants k12 and kin lead to matches of simulated and experimentalcurrentslike that shown in Figure 2. Thecomparison is quite satisfactory and, for the experiment shown, yields the rate constants kl2 = (1.2 f 0.2) X lo7 M-I s-' and kin = (1.3 f 0.02) X lo2s-I in pH 8.5 TRIS with 0.1 M C1-. The uncertainty cited is that of matching the simulation to the data set. Spectrophotometric Rate Measurements. [Co(terpy)2I3+exhibits an absorbance band at 352 nm, at which the absorbances of sulfite, sulfite oxidase, and [Co(tcrpy)2l2+are not significant. Consumption of [ C ~ ( t e r p y ) ~by ] ~ reactions + 6 and 7 can thus be monitored using the decrease in 352-nm absorbance occurring uponadditionofsulfitetoamixtureofenzymeand [Co(terpy)2]3+. The reaction velocity is measured as an initial rate to avoid the effects of depletion of sulfite or of [ C ~ ( t e r p y ) ~ ] ~ + . Figure 3A shows the reaction velocity data obtained at varied [C0(terpy)~]3+concentrations and constant enzyme and sulfite concentrations. The increase in reaction velocity is linear at low [ C ~ ( t e r p y ) ~but ] ~ +folds over at high concentrations. The effect is analogous to that in Figure 2 and is again interpreted as onset of rate control by the intra-enzymereaction, reaction 7. Analysis of spectrophotometricreaction velocity data according to eq 10 is shown in Figure 3B,for an experiment similar to Figure 3A but conducted at higher [Co(terpy)2I3+concentrations. The reciprocal plot is linear with a substantial intercept; its leastsquares slope and intercept, using q 10, yield valuesof k l z= (9.4 f 2.6) X 106 M-l s-I and kin = (1.1 0.5) X lo2 s-I. These results, in pH 8.5 TRIS with 0.1 M C1-, are in excellent agreement with those from Figure 2 using mediated electrocatalysis. Reaction Kinetics for kl2 and 4.M a Function of Mediator. If the reaction rate limitation seen in Figures 2 and 3 is correctly
*
8.00-05
1.21+07
0
Y
5
I
where Cm,* is the bulk mediator concentration. Inspection of limiting electrocatalytic currents measured as a function of mediator concentration (Figure 2) reveals, however, a nonlinear behavior: a flattening out of current (e.g., mediatorheme reaction rate) a t high concentration. The nonlinearity is consistent with electrocatalytic regeneration of [Co(terpy)212+becoming limited by a reaction step additional to that implicit in eq 1 1,such as the intra-enzyme electron transfer of reaction 7. To analyze data like that in Figure 2, the Fick's law diffusion quation for the oxidized form of the mediator is modified according to the reaction velocity in q 10 to yield
6.00-05
Conc. Co(terpy),S' (M)
Conc. Co(terpy),2' (M) Figure 2. [Co(ttrpy)2l2+ concentration dependency of steady-state
4.01-05
f
1.0e+07
0 0
c
8.01+06
t
B
/*
/
1
I I
s
5
4.01+06 O.Oe+OO
2.5@+04 5.0@+04 7.5@+04 1.00+05
1/Cone. Co(terpy)F (1 /M) Figure 3. [Co(tcrpy)#+ concentration dependency of spectrophotometrically determined initial reaction velocity: (A) with 1.24 nM batch A enzyme, 0.43 mM sulfite; (B) with 2.23 nM batch B enzyme and 3.39 mM sulfite.
TABLE I: Rate Constants for the Electron-Transfer Cross-Reaction of the sulfite Oxidase Heme Site with Different Mediators, in 20 m M Phosphate Buffer, pH 7.0, Using Batch C Enzyme mediator '?l k22,b M-'s-' kl2: M-I s-I k,,,'s-I [Co( terpy)2]2+/3+ ECHEM 0.40 SPEC cyt c 0.083 [Co(bpy),l2+/3+ 0.084
400 5.4 X 18
(7.0 i 3.4) x (1.1 i 0.1) x 10) (9.7 & 4.3) X (4.4 & 0.3) X
107 107 lo* IO5
72 7 94 i 28 87 4 89 & 13
*
Formal potentials for mediators, Yvs Ag/AgC1(0.197 V vs NHE).6 Electron self-exchange rate constants for mediators.6 This study with [sulfite oxidase] = 0.72 pM, [sulfite] = 6.3 mM. Tolerances are 90% confidence intervals; N - 1 degrees of freedom.
interpreted as representing intervention by the intra-enzyme reaction, reaction 7,then values of kinshould not depend on the particular mediator-oxidant employed. Values of k12 should, on the other hand, depend on the mediator through its formal potential, which governs the free energy6 of its reaction with the heme and, through k22, the mediator's electron self-exchange rate constant. The results of an examination of the precedingpoint are given in Table I, using three different mediator systems that have similar formal potentials ( E O ' ) but values of k22 that differ by a range of 3 X 103-fold. Thesemediators are known6toundergo electrontransfer cross-reaction at the heme site of the enzyme rather than at the Mo site. The mediator-heme cross-reactionrate constants k12vary widely and in the same order as k22, whereas the results for kin are, within experimental uncertainty, all the same, with a mean of 86 f 11 s-l. These results support the analysis of the fast-reaction rate limitation as a mediator-independent, intraenzyme oxidation of reduced enzyme heme site, presumably by reaction with the enzyme Mo site as in reaction 7, and argue against interpretation of the k,n step as a mediator+nzyme association process which presumably would be mediatordependent.
Reaction Kinetics of Sulfite Oxidase 1.8e-06
The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1697
I
0.0e+00
1.5e-04
I
91
I
4 ' 0.00
3.0s-04
Conc. Co(phen),2+(M) Figure 4. [Co(phen)3l2+ concentration dependency of steady-state electrocatalytic currents. ( 0 )Experimental currents from potential step measurements in 20 mM TRIS/O.l M KCI buffer, pH 7.5, 0.72 pM batch C enzyme, 6.3 mM sulfite; (+) simulated current based on kl2 = 8.0 X 107 M-1 s-I and k,, = 320 s-1.
We have obtained9 results analogous to those in Table I (mediator-independent k,, and -dependent klz) using electrogenerated organic mediator oxidants, rat liver sulfite oxidase, and pH 7.5 20 mM TRIS/O.l M KCl buffer. The k,,in rat liver sulfite oxidase (ca. 10 s-I) is lower than the values observed with chicken liver enzyme (see later Tables I11 and IV). As noted earlier, we found in a previous study6 (in which the limitingeffectsof kinwere neglected) that the freeenergy-reaction rate relation within a series of structurally similar, electrogencrated [Co(Mexphen)3]3+mediator-oxidants at high reaction rates began to deviate from classical Marcus theory predictions. These experiments have now been repeated using the improved kinetic interpretation developed above and choosing concentration conditions that accentuate fast enzyme turnover rates. Figure 4 shows currents obtained with electrogenerated [C~(phen)~],+, which has thelargest reaction freeenergy inthe [C0(Me,phtn)~J3+ mediator4xidant series and which exhibited6 a deviation from the Marcus free-energy relation when the k12 rate constants had been obtained using eq 1 1. These data, clearly showing effects like that observed with [Co(terpy)z13+in Figure 2, wereanalyzed by matches to current-concentration plots calculated from eq 12. The results of this new analysis for the [Co(Me,phen)3I3+ mediator-oxidant series are presented in Table 11. The Table I1 results for the mediator-heme cross-reaction rate constant klz show, at both pH 7.5 and 8.5, a steady increase with the potential ( E O ' ) of the [C~(Me,phen)j]~+/~+ couple. The k12 values from classicaltheory6 are expected to depend on the electron self-exchange rate constants of the enzyme heme site ( k l l ) and mediator (k22)and on the reaction free energy as reflected in the differences between formal potentialsof the [C0(Me,phen)~]3+/2+ couple (Table 11) and that of the enzyme heme site (-0.1 13 V vs. Ag/AgCl),IO according to the relation log k,, = 0.5 log (kllkZz) + 8.45hE0'[1 where
x=
[0.2367 log
+x u o ' I
(13)
(-->]-Ik,Ik22
z2
and Z,the collision-limited rate constant, is estimated6 as 1.8 X lolo M-1 s-1. Figure 5 shows data from Table I1 iterated (for x ) to produce a best linear fit to q 13. The least-squares slopes, 8.99 at pH 8.5 (curve A) and 8.89 at pH 7.5 (curve B), are close to theoretical, 8.45. Figure 5C shows, for comparison, the rate constants from the previous study,6 which was done at pH 7.5 and which neglected the intra-enzyme reaction (kin). The k12 rate constants obtained in the present study (Table 11) are uniformly larger than the previous values due to use of a higher activity enzyme preparation. The foldover seen in the previous results at high AEO' (Figure 5C) is absent in Figure SA,B since
1
J 0.06
0.12
0.18
0.30
0.24
AEo'(1 +XAEO')
FigureS. k12dataforhemesitecross-rtactions withcobalt phenanthrolines numbered as in Table I1 plotted according to eq 13; error bars = 90% confidence intervals of k12 estimations from numerical matches as in Figure 4; line shown is linear least-squares regression. (A) pH 8.5; (B) pH 7.5; (C) data from ref 6,neglecting kin,and with a less active enzyme. 10
1
1 I
P
5 ' 0.00
I 0.06
0.12
0.18
0.30
0.24
AE"
8
9
1
6
5 0.00
0.06
0.12
0.18
0.24
0.30
AEO' Figure 6. k I 2data for heme site cross-reactions with mediator in Tables I and I1 (pH 8.5); where p i n t 7 is [Co(terpy)#+; 8 is ferricytochrome c; 9 is [C~(bpy)~]f+;vs differences in hemeand mediator formal potentials;
error bars = 90% confidence intervals of constant estimations from numerical fitting as in Figures 2 and 4, line shown is linear least-squares regression.
the limiting effects of kin are properly accounted for there in the kinetic analysis. That is, the data of Table I1 now show accord with the classical rate-free-energy equation (13) over the entire range of [Co(Me,phen)~]~+/~+ reaction rates. The electron self-exchange rate constants k22 within the [C~(Me,phen)~]-'+/*+ series arc essentially invariant in the eq 13-Figure 5 analysis. Figure 6A shows that the k12data for the three mediators in Table I, whose kz2values are widely different, do not agree well in a log klrhEO' plot with the Table I1 [C0(Me,phen)3]~+/~+ k12data, but Figure 6B shows that when the different k22 values are taken into account by plotting (log k12- 0.5 log k22) against Uo', the results for electrogenerated [Co(terpy)213+and ferricytochrome c now agree rather well. The rate constant for [Co(bpy)3J3+continues, however, to lie below the correlation line. Table I1 also presents results from the [Co(Me,ph~n)~]3+/2+ series for k,,,derived from plots like that in Figure 4. In general,
Yang et al.
1698 The Journal of Physical Chemisrry, Vol. 97, No. 8, 1993
TABLE II: Rate Constants for the Electron-Transfer Cross-Reaction of the Sulfite Oxidase H e w Site with Electrogcaanted [Co(Me#hen)3r+ Mediators, in 20 m M TRIS Buffer with 0.1 M KCI, Using Batch C Enzyme
1.
mediator complex [C~(phen),]~+/~+ [Co(S-Me-phen)~l~+/~+ [C0(4-Mephen)j]~+/~+ [C0(5,6-Me2phen)3]~+/~~ [Co(4,7-Me2phen)312+/-'+
F
2. 3. 4. 5. 6. [C0(3,4,7,8-Me4phen)3]~+/~+
a
0.152 0.096 0.039 0.014 -0.052 -0.075
k22.6
M-'S-' 45 45 45 26 7 45
k12.C M-1 s-I pH 7.5 pH 8.5 (1.2 f 0.5) X IO* (8.0 f 2.0) X IO7 (1.5f0.5)X107 (5.0f1.0)X107 (1.5 0.5) x 107 (2.5 f 1.0) x 107 (5.0 0.7) X IO6 (1.0 f 0.3) X lo7 (4.0 f 1.5) X IO6 (3.0 f 1.3) X IO6 (2.0 f 0.3) X lo6 (8.0 f 4.0) X IOs
kin: s-' pH 7.5 pH 8.5 320 f 30 365 f 40 180f20 500k50 400 40 400 40 200 f 20 320 f 30 250 f 30 350 f 40 7 0 * 10 1Of6
0 Redox potentialsof [Co(Me,phen)3]ft mediators, Vvs Ag/AgCI (0.197 Vvs NHE).6 Electronself-exchange rateconstants for [Co(Me,phen)313+ mediators? C This work, with [sulfite oxidase] = 0.72 pM, [sulfite] = 6.3 mM. Tolerances are 90% confidence intervals; N - 1 degrees of freedom.
TABLE HI: Buffer Dependency of Rate Constants for the Reduction of [Co(terpy)#+ by Sulfite-Saturated SuVte Oxidase (Batch B) buffer 20 m M TRIS, 0.1 M KCI, pH 8.5 SPEC" ECHEMb 20 mM phosphate, pH 8.5 SPECd ECHEMb 20 mM phosphate, pH 7.0 SPECd ECHEMb
k,,,'s-I 110 f 50
131 f 2
k12: M-'
S-'
(9.4 2.6) X IO6 (1.2 f 0.2) X IO7
97 f 31 70 & 8
(8.2 f 0.7) X IO6 (2.3 f 0.3) X IO7
35 f 8 24 f 2
(4.4 f 0.5) X IO6 (1.6 f 0.2) X IO7
Spectrophotometricexperimentwith batch Benyme, [sulfiteoxidase]
= 2.2 nM, [sulfite] = 3.5 mM, [Co(terpy)#+ = 10-50 pM. * Catalytic
voltammetric experiment with batch B enzyme, [sulfite oxidase] = 0.68 pM, [sulfite] = 6.3 mM, [Co(terpy)2I3+ = 8-80 pM. Tolerances are 90%confidence intervals;N - 1 degrees of freedom. Spectrophotometric experiment with batch B enzyme, [sulfite oxidase] = 7.2 nM, [sulfite] = 3.4 mM, [Co(terpy)J3+ = 2-20 pM.
k,,changes little if at all within the series of complexes. (The result for the slowest reacting complex is less reliable, since the kinetic limitation imposed by k,,when k12is small is weak and more difficult to measure.) A previous study4 of k,,,in which laser flash photolysis was employed to initiate the Mo-heme reaction, gave a value of 310 s-I for the Mo heme electron transfer. The pH 7.0 phosphate buffer employed in Table I differs from that used in the earlier study: lacking EDTA and chloride. The result for k,,in Table 1(86 s-I) is smaller than the previous4result by only ca. 4-fold, which given thedifference in buffer and the substantial differences in methodology can be taken as reasonably good agreement. The k,,results in Table 11, taken in TRIS buffer containing chloride, are closer to that of the earlier4 study. However, as we show below, k,,values in TRIS buffer are larger than those in pH 7 phosphate buffer, so the Table I1 TRIS buffer data should not be compared to the previous4 k,,result. Reaction Kinetica for k12 and kin as a Function of Buffer, Electrolyte,and Ionic Strength. Table I11 reports electrocatalytic and spectrophotometricexperimentsexaminingthe effect of buffer system and pH on the k12 and kin rate constants using [Co( t e r ~ y ) ~mediator. ]~+ The effect of pH on k12and k,,in TRIS/ KCl buffer was shown in Table 11. These results show that the intra-enzyme electron-transfer rate constant k,,(a) is, at constant pH, smaller in phosphate than in chloride buffer (Table 111) and (b) in both TRIS/KCl and phosphate buffers decreases with decreasing pH (Tables I1 and 111). In Table 111, both the electrocatalytic and spectrophotometric k,, results show these trends. As for the heme-mediator k12rate constant, the results show it also decreases with decreasing pH (Tables I1 and 111) but at pH 8.5 is about the same in phosphate and in TRIS buffer (Table 111). The optimum pH for sulfite oxidase activity has been reported" as pH 8.5, which is consistent with the pH dependencies of k12 and k,,. Redox potentials of the heme Fe(III/II), Mo(IV/V), and Mo(V/VI) sites of sulfite oxidase have recently been reported as functions of buffer composition and pH.l2>13 The data indicate
that as pH decreases, heme Fe(I1) is a less strong reductant and the heme-mediator electron transfer consequently less favored. On reaction free-energy grounds, then, the observed pH dependency of k12 is in the anticipated direction. Both chloride and phosphate form complexes at the Mo site of sulfite oxidase,12J4 so rate effects in buffers containing these ions (Table 111) are in the case of kin unsurprising. Table IV shows the kinetic results of varying the concentration of KCl in pH 7.5 TRIS buffer, using electrogenerated [Co(phen)3I3+as the mediator-xidant. The decrease in the electron-transfercrossreaction rate constant k12 with increasing [KCl] is analyzed (below) as an ionic strength effect. The appreciable change in the intra-enzyme rate constant kin is considered next. The studiesl2J3 of sulfite oxidase active site formal potentials report that chloride complexation at the Mo site produces substantial changes in the Mo site formal potential; that for the heme site is relatively unaffected. Chloride binds more strongly to the Mo(1V) than to theMo(V) state,soitseffect on thepotential of the Mo(V/IV) couple is larger.l* The chloride concentration dependency of the Mo (V/IV) formal potential ( E j ) isI2 given by
-
where E 3 O is the Mo(V/IV) standard potential (-0.233 V vs NHE), 4 , the dissociation constantls for the Mo(V)-Cl state, is 4.0 X M2, and Ks,the dissociation constant for the Mo(IV)-Cl state, is 1.2 X 10-11 M2. We examine the change in kin with chloride concentration in Table IV with two simple models, assigningin one the rate change to a chloride-induced reaction free-energy change via the potential of the Mo(IV/V) site and in the other to a smaller rate constant for electron transfers from Mo sites complexed by chloride. Considering the first model, values of the Mo(V/IV) potentials (E3) and the corresponding Mo(V/IV)-heme reactant potential differences ( M )and reaction free energies (AC)were calculated at different chloride concentrations as shown in Table IV (middle columns). On reaction free-energy grounds, kin should be proportional to exp(-AG/2RT) and Table IV shows that the ratios of these quantities at different chloride concentrations are indeed fairly constant. That is, a simple model of changing the Mo(V/IV) potential and thus the reaction free energy is consistent with the experimental behavior of kin. The second model presumes that chloride inhibits Mo site reactivity more specifically,such as by a small Mo-heme electrontransfer rate constant for chloride-bound Mo sites or by retarding an associated step in the Mo site cycle such as regeneration of the Mo-oxo bonding lost after sulfite reacti011.I~If we assume that only Mo sites uncomplexed by chloride are reactive in intraenzyme electron transfer, a linear relation between the experimentally observed kinand the concentration of uncomplexed sulfite oxidase Mo sites becomes predicted. Table IV (right-hand
The Journal of Physical Chemistry, Vol. 97, No. 8, 1993 1699
Reaction Kinetics of Sulfite Oxidase
TABLE Iv: chloride Concentration Dependency of Electron-Transfer Rite Constants Using [Co(pI1en)3]~+ as Electrogenemted Mediator-Oxidmt. in 20 mM TRIS/KCL DH7.5 Buffer [Cl], M 0.01 0.1 0.2 0.3 0.4 0.5
&12,'
M-'
S-'
(1.0 f 0.1) X (2.0 f 0.2) X (4.5 f 0.5) X (4.0 f 0.5) X (2.0 f 0.1) X (1.5 f 0.2) X
&i",O
IO9
IO8 10'
10' lo7 lo7
s-I
600 f 20 350 f 20 255 f 10 I80 t 10 150 f 10 130 f 10
Elb
AEr
AG,d kJ/mol
&in/exp(-AG/2RT)
[free Mol: nM
ki./([free Mol x 109)
-0,150 -0.105 -0.097 -0,093 -0.091 -0.090
0.234 0.189 0.181 0.177 0.175 0.174
-22.59 -18.25 -17.43 -17.09 -16.90 -16.78
6.46 9.02 7.73 5.84 5.05
26.44 2.74 1.37 0.91 0.69
4.49
0.55
22.7 127.7 186.1 197.8 217.4 236.4
0 This work with batch C enzyme, [sulfiteoxidase] = 0.72 pM, [sulfite] = 6.3 mM. Tolerances are 90% confidence intervals;N- 1 degrees of freedom. Formal potential of Mo(V/IV) calculated from eq 15 (YvsNHE). C Difference in formal potentials of Mo(V/IV) and heme (takinglo heme redox potential as +0.084 V vs NHE). Reaction free energy between Mo(V/IV) and heme calculated from AG = -nFAE. Calculated concentration of sulfite oxidase Mo uncomplexed by CI-.
b
-
6.0e-07I
.--
t
n 0
J -
-
t
C
.-0 0
m m -
14
'
-2.0
I -1.0
E
1
o
I
.
I
1.5e-07
'
o.oe+Oo 0.0e+00
I 4.0e-05 8.0e-05
1.2e-04
1.6e-04
0.0
ionic str. function Figure 7. log plot of [Co(phen)J3+ mediator &12 data from Table IV against ionic strength function defined in eq 16; line shown is linear least-squares regression.
columns) shows uncomplexed Mo site concentrationscalculated from the dissociation constant KSand the ratio kin/[free Mol. While the ratios from 0.1 to 0.5 M [Cl-] are reasonably similar, there is a noticeabletrend; also, the ratio at 0.01 M [Cl-] is quite different. While this model thus seems to be less satisfactory, we believe a difference in chloride-bound Mo reactivity remains a possibility for further study. The decrease in k12 fqr [Co(phen)J3+ in Table IV with increasing [KCl] is in the direction expected for increased screening of a favorable electrostatic work term at higher ionic strength. Previous data6for this mediator were affected at lower ionicstrengthby neglect of ki,limitations. Using theionicstrength functionflp) of Wherland and Gray16
(16) where K = 0.329pl12, Re,, and Rmd are effective heme site and mediator sizes, and zm& is the mediator charge, Figure 7 shows a least-squares fit ofthek12datainTableIVagainstflp)assuming Re,, = 5 A. The intercept yields an estimate of k12= 5.58 X 106 M-l s-I at infinite ionic strength. The plot's linearity is relatively insensitive to the assumed value of Re,, over 5-10 A; the slopes of plots for this range of Re,, yield estimates of charges (zenZ)of -3 to -5 for the heme site. Values of z,,, of -7 to -10 were estimatedbusingrates from electrogenerated[Co(bpy)3l3+.This approach to estimating zenris, while obviously not very exacting, supportive of the previous conclusion6 that the sulfite oxidase heme site bears a negative charge that strongly affects electrontransfer cross-reaction rates with ionic mediator-oxidants. We note in passing that the Table I11 and Figures 2 and 3 measurementsat pH8.5TRIS,O.l MCl-weremadewithenzyme preparation batch B, whereas Tables I, 11, and IV represent data taken with a somewhat more active enzyme preparation (batch C, reductase activity toward ferricyanide is larger by ca. 2-fold; see Experimental Section). We see that the k12 and kin rate constants in pH 7.0 phosphate buffer are both 2-3-fold larger in Table I than those in Table 111. We did not normalize for the
Conc. Co/Zn(terpy),2' (M) Figure 8. Spectrophotometric inhibition test of [Co(terpy)2l2+and [Zn(terpy)212+.With 1.24 nM batch B enzyme, 1.7 mM sulfite and 24.2 pM [Co(terpy)2]'+, in 20 mM TRIS/O.l M KCI buffer, pH 8.5 (A) [Co(terpy)2I2+;(B) [Zn(terpy)2I2+.
differencein ferricyanidereductase activity for these two enzyme preparations, since the calibrant ferricyanide reacts at least to some extent' with the Mo site and an enzyme activity correction is thus not straightforward. Inhibiting Kinetic Effect of [C~(terpy)~]~+. The spectrophotometric results discussed above were obtained using solutions containing initially only [Co(terpy)2]3+ and no [Co(terpy)2]2+. Adding [ C ~ ( t e r p y ) ~to ] ~the + initial solution depresses the initial reaction velocity of [C0(terpy)2]~+as shown in Figure 8A. This result suggests that the reduced mediator [C0(terpy)~]2+can inhibit the sulfite oxidase reaction. Considering the possibility of strong complexation between the mediator cation and the negatively charged patch around the heme site: we added [Zn(ter~y)~]2+ as a nonelectron-transfer-activesurrogate for [Co(terpy)2I2+. Figure 8B shows that the enzyme reaction rate is not changed by addition of [Zn(terpy)2I2+;reaction inhibition by competitive heme-site complexation seems to be ruled out by the result, as are slow association reaction steps between mediator and mediator. A plausible origin of the inhibiting effect of added [Co( t e r p ~ ) ~is] ~promotionof + the back-reactionof the cross-electron transfer, Le., that of heme Fe(II1) with [Co(terpy)2I2+.A hememediator cross-reactionequilibrium constant of 10 would serve1' to account for the inhibition seen in Figure 8A. The actual reaction equilibriumconstant, estimated'* for reaction 6 from literature4fj redox potentials, is somewhat larger, 3.7 X lo2, so some other factor may also be involved. Finally, we note that although solutions of [ C o ( t ~ r p y ) ~are ]~+ used as the source of [Co(terpy)2]3+ in the mediated electrocatalysis experiments, an inhibiting effect of [Co(terpy)212+is not expected during electrocatalysis. This is because, in the steadystate reaction layer of solution near the electrode surface, most of the [ C ~ ( t e r p y ) ~ ]mediator ~+ has been electrochemically converted to the [Co(terpy)2I3+form.
Acknowledgment. This research was supported in part by a grant from the National Science Foundation. We are grateful
1700 The Journal of Physical Chemistry, Vol. 97, No. 8, 1993
to J. L. Johnson and K. V. Rajagopalan of the Department of Biochemistry, Duke Univenity, Durham, NC, who have provided technical advice and purified enzyme during this project.
References md Notes ( I ) Rajagoplan, K. V. In Molybdenum and Molybdenum-Containing Press: Oxford, U.K., 1980;pp Enrvmes: Counhlan. M.. Ed.:Pergammon .. 241-272. (2) Kramer, S. P.;Johnson, J. L.; Ribeiro, A. A.; Millington, S. S.; Rajagopalan, K. V. J. Biol. Chem. 1987, 262, 16357-16363. (3) Cohen, H.J.; Fridovich, I. J . Biol. Chem. 1971,246,359-366.367-
-171 . -.
(4) Kipke, C. A.; Cusanovich, M.A,; Tollin,G.; Sunde, R. A.; Enemark, J. H.Biochemistry 1988,27, 2918-2926. (5) Coury, L.A.;Oliver, 8.N.; Egekeze, J. O.;Sosnoff,C. S.;Brumfield, J. C.; Buck, R. P.; Murray, R. W. Anal. Chem. 1990,62,452-458. (6) Coury, L.A.; Murray, R. W.; Johnson, J. L.;Rajagopalan, K. V. J . Phys. Chem. 1991,95,60344040. (7) (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 . Biol. Chem. 1972,249,50465055.(c) Johnson, J. L.; Rajagopalan, K. V. J . Biol. Chem. 1917,252,2017-2025.(d) Cohen, H.J.; Fridovich, 1. J. Biol. Chem. 1971,246, 359-366. (8) Bard, A. J.; Faulkner, L. R.Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980. (9) Coury, L. A.; Yang, L.; Murray, R. W. Anal. Chem., submitted. (10) Cramcr, S.P.; Gray, H. B.;Scott, N. S.; Barber, M.; Rajagopalan, K. V. In Molybdenum Chemistry of BiologicalSignificance; Newton, W . E.,
Yang et al. Otsuka, S.,Eds; Plenum: New York, 1979; pp 157-168. ( 1 1 ) MacLcod, R. M.; Farkas, W.; Fridovich, I.; Handler, P. J. Biol. Chem. 1961,236, 1841. (12) Spencc, J. R.; Kipke, C. A.; Enemark, J. H.; Sunde. R. A. Inorg. Chem. 1991, 30, 301 1-3015. (13) Kipke, C. A. Physical Studies and Synthetic Modeling of the Molybdenum-Containing Enzyme Sulfite Oxidase. Ph.D. Thesis, The University of Arizona, 1988. (14) Kessler, D. L.;Johnson, J. L.; Cohen, H. J.; Rajagopalan, K. V. Biochim, Biophys. Acta 1974,334,86-96, (15) Bray, R. C.; Gutteridge, S.;Lamy, M. T.; Wilkinson, R. Biochem. J . 1982,211, 227. (16) (a) Wherland, S.;Gray, H. B. In Biological Aspects of Inorganic Chemistry; Addison, A. W., Cullen, W. R., Dolphin, D., James, B. R., Eds.; Wiley: New York, 1977;pp 289-368. (b) Wherland, S.;Gray, H. B. h o c . Natl. Acad. Sei. U.S.A. 1976,73,2950-2954. (1 7) When the back-reaction of the mediator-hemecross-electron transfer ~ oEnQx, with K12 = k l ~ / k - ~ 2 , is considered, Medox + EnzREo * M e d ~ + the expression for reaction velocity becomes 1/Vel = (kl~[Medox]+ k,")! (k,nk12[MedoxlCm') + k - ~ z [ M C d ~ ~ ~ ] / ( k , ~ k ~ z [ M e dwhere o x ] c ~k-12 " ~IS* ) , the rate constant for the heme Fe(III)-[C0(terpy)2]~+back-reaction. A backreaction rate constant k-12 = 1.0 X lo6 M-' s-' and an equilibrium constant K12= 10 are estimated from the slope of a l/Vel vs [ M ~ ~ R E plot D ]of the Figure 8A data, taking rate constants k,, and k12 as given in Table 111. (18)K I * =exp[(nF/RT)hE], where Misthedifferenceinreactantredox potentials. Thepotentialsofheme Fe(II/III) and [C~(terpy)~]~+/)+ are taken6 as -0.1 13 and 0.040V vs Ag/AgCl, respectively.
