Reactivity comparison of iron(II) reductant with type 1 copper(II) in native and type 2 copper-depleted Rhus vernicifera laccase

Biochimica et Biophysica Acta, 791 (1984) 112-116

112

Elsevier

BBA Report BBA 30083

REACTIVITY COMPARISONS OF IRON(Ii) REDUCTANTS WITH TYPE 1 COPPER(II) IN NATIVE AND TYPE 2 COPPER-DEPLETED R H U S VERNICIFERA LACCASE R O B E R T A. H O L W E R D A *, H A E S U N K A N G BAEK, R. MAX W Y N N and D A V I D B. K N A F F

Department of Chemistry, Texas Tech Unioersity, Lubbock, TX 79409 (U.S.A.) (Received August 1st, 1984)

Key words: Laccase reduction; Electron transfer rate; Hydroxyethyl ferrocene," (R. vernictfera)

Rate constants and activation parameters are compared for the reductions of native and type 2 copper-depleted (T2D) Rhus vernlcifera laccase type 1 Cu(ll) by hydroxyethylferrocene, Fe(CN) 4- and Fe(EDTA) 2- . Oxidation of Fe(CN) 4- (k(25°C) -- 1.45 • 102 M - i . s - i, pH 7, I = 0.5 M) by T2D iaccase blue copper is an order of magnitude faster than the corresponding native enzyme rate, and a type 2 Cu(II)-Fe(CN) 4interaction is shown to be responsible for complex kinetic behavior in the reduction of native laccase. Activation parameters ( A H ~, A S * ) confirm the presence of a large conformational rearrangement harrier in the electron transfer pathway to laccase type 1 Cu(ll), as compared with other blue copper proteins. A systematic compensation pattern between AH* and AS ~ in laccase reductions by Fe(ll) redox agents suggests a common mechanism, with considerable flexibility in activation requirements, dependent upon the hydrophilicity of the electron donor.

The availability of Rhus oernic~[era laccase specifically depleted of the type 2 copper atom has greatly aided the assignment of enzymatic roles to this solvent-accessible, EPR-detectable site [1]. While removal of type 2 Cu(II) has no effect on the 'blue' (type 1) copper reduction potential, laccase-polyphenol binding in the pathway leading to type 1 Cu(II) reduction is severely retarded by this modification [1], confirming the substratebinding role of type 2 Cu(II) [2,3]. The electron transfer reactivity of tree laccase type 1 Cu(II) with both organic and inorganic reductants is notably lower than the analogous rates characteristic of the fungal enzyme and blue copper proteins containing only type 1 sites (stellacyanin, plastocyanin, azurin) [4]. There is some indication, however, that the intrinsic electron transfer reactivity of laccase type 1 copper increases considerably upon removal of the type 2 copper atom [1]. We * To whom reprint requests should be addressed. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

report here rate parameter comparisons for the reduction of native and T2D laccase blue copper by iron(II) redox agents, including Fe(CN)46- , hydroxyethyl ferrocene and Fe(EDTA) 2- . Our goals are: (1) To understand better the effect of type 2 copper removal on type 1 Cu(II) reduction rates; (2) to clarify the mechanistic basis for the low redox reactivity of laccase type 1 copper, as compared with other blue copper proteins; and (3) to determine whether ligation of type 2 copper is the source of complex kinetic behavior reported for the Fe(CN)4--native laccase type 1 Cu(II) redox reaction [5]. Such a type 2 copper-ferrocyanide interaction is suggested by the significant dependence of laccase E ° values on the concentration of the Fe(CN) 3-/4- mediator couple [6]. Emphasis is placed here on the activation enthalpy and entropy, as AH¢, AS¢ values offer a deeper mechanistic insight than simple rate constant comparisons [7,8]. All rate measurements were carried out on I =

113

0.5 M (phosphate), pH 7 solutions. Redox agents, native and T2D lactase were obtained and purified as previously described [ 1,2,5]. Anaerobic kinetic measurements at 614 nm were performed on a Durrum D-110 stopped flow apparatus [1,2], using pseudo-first-order conditions for the metalloenzyme (approx. 10 PM). Rate data were transmitted directly to an Apple II plus computer, stored on floppy disk, and quantitatively interpreted through a modified Interactive Microware interface (unpublished results). Reported first-order rate constants ( kobs), calculated as least-squares slopes of ln( A, - A,) vs. time plots [1,2], generally are the mean of 3-5 trials. A simple one-step mechanism (Eqn. 1) for the approach of Fe(CN)zand lactase type 1 Cu(I1) to redox equilibrium would give rise to the rate law of Eqn. 2 [5]. Fe(CN);-

+T~CU(II)~~

Fe(CN);-

+TlCu(I)

I k ohs= k, [Fan-]

+ k_, [ F+cN):-]

(1)

(2)

Our kinetic results on the reduction of T2D, (reduced type 3 site) lactase type 1 Cu(I1) by Fe(CN)z(Table I) are entirely consistent with this expectation. Thus, in marked contrast to the apparent mixed first- and second-order Fe(CN)zdependence characteristic of the native enzyme [5], reduction of Tl Cu(I1) in T2D lactase is clearly first order with respect to Fe(CN)zover the 0.25-10.0 mM interval (k,(25”C) = (1.45 L- 0.03). lo2 M-’ . SC’). Oxidation of Fe(CN)zby T2D lactase blue copper is an order of magnitude faster than the corresponding native enzyme rate under the same conditions, supporting our previous conclusion that T2D Tl Cu(I1) enjoys an appreciable redox reactivity advantage. profile at constant The kobs vs. [Fe(CN)i-] [Fe(CN);f-] = 10.0 mM also is linear, yielding k_, = (3.9 f 0.3). 10’ M-’ - SC’ (25’C). An apparent equilibrium constant (K,) for reaction (1) of 3.7 (25YZ) may be calculated as k/k_, from the forward and reverse rate constants. Knowing that E” (Fe(CN)z-/4-) = + 435 mV vs. standard hydrogen electrode under our conditions [9], the T2D Tl Cu(II,I) reduction potential implied by this apparent equilibrium constant is +470 mV, sub-

TABLE

I

OBSERVED RATE CONSTANTS FOR THE REDOX REACTIONS OF Fe(CN)z-/‘WITH T2D LACCASE TYPE 1 Cu(I1, I) pH 7, I = 0.5 M (phosphate), Rate constants are the mean of at least three trials, with a typical uncertainty of f 5%.

[WCN)i- 1

IFe(CN)i- 1 (mM)

Temperature (“C)

kbs (s-l)

(mM) 0.25 0.50 1.00 2.50 5.00 7.50 10.0 10.0 10.0 10.0 10.0 10.0 1.063 1.063 1.063 1.063 1.063

0

25.0

0.0503 0.0980 0.167 0.353 0.706 1.05 1.50 1.48 1.51 1.58 1.64 1.75 0.0256 0.0388 0.0852 0.343 0.639

0 0 0 0 0 0

0.50 1.00 2.50 5.00 7.50 0 0 0 0 0

10.9 14.8 20.3 30.5 36.2

stantially more positive than the true value of + 429 mV [l]. Overall 614 nm absorbance changes (AA,,,) at constant lactase and Fe(CN)zconcentration decrease with increasing [Fe(CN)i-1, as expected for a redox reaction in which the oxidant is not fully (over 99%) consumed at equilibrium. Since only Tl Cu(I1) (with extinction coefficient E,,) absorbs significantly at 614 nm, Eqn. 3 describes the dependence of AA,,, on K, and hexacyanoferrate concentrations expected from mechanism (1); I is the spectrophotometric pathlength:

AA 614 = ~,,l[laccase]

K, [ WCV~-] [ Fe(CN)i-]

+ K, [ Fe(CN$]

(3)

An adequate non-linear least square fit of AA,,,, [Fe(CN)z-/3-] points to Eqn. 3 was achieved with K, = 1.4 + 0.2, consistent with an E” Tl Cu(I1, I) value (+444 mV) in much better agreement with the results of spectroelectrochemical titrations using hydroxyethyl ferrocene as a mediator [l]. While the discrepancy between kinetic and spec-

114

trophotometric K e calculations appears, at first glance, to be a violation of the microscopic reversibility principle, no contradiction would exist if appreciable amounts of hexacyanoferrate ions remain complexed to laccase at equilibrium. Thus, metalloprotein reduction potentials measured in the presence of an inorganic mediator reflect, in part, the extent of mediator-protein binding in both oxidized and reduced forms [10]. Such strong complexation of metalloproteins by Fe(CN) 4-/3is well documented [6,10-16]. Our present results therefore underscore the importance of using noncomplexing mediators in metalloprotein E ° determinations [1]. The temperature dependence of the kinetics of the Fe(cN)a--T2D T1 Cu(II) reaction and of the hydroxyethyl ferrocene reductions of both native and T2D laccase are summarized in Tables I and II, respectively. For the purpose of constructing Eyring plots of ln(k/T) vs. I/T, second-order rate constants were calculated as kobs/[reductant] in runs where no Fe(III) product was present initially; a first-order hydroxyethyl ferrocene dependence was established previously at 25.0°C [1]. Activation parameters derived from the leastsquares analysis of these Eyring plots are compared in Table III with analogous parameters reported for other blue copper protein reactions with Fe(II, III) redox agents. The modest reactivity advantage of T2D over native laccase in the oxidation of hydroxyethyl ferrocene may be entirely attributed to the AS* term (more positive by 3 e.u. *).

A mechanism involving reversible anation of type 2 copper by Fe(CN)64- followed by reductive attack of a second ferrocyanide ion on the blue copper center best fits kinetic data for the reduction of native laccase [5]. Dramatic rate enhancements at high [Fe(CN)64- ]/[laccase] ratios implied that a ferrocyanide-enzyme interaction strongly activates type 1 "Cu(II) towards electron transfer. The simple first-order Fe(CN)64- dependence in the Fe(CN)64--T2D T1 Cu(II) reaction, coupled with Fe(CN)4--induced perturbations in the type 2 Cu(II) EPR signal [6], clearly confirms this mechanism. A similar interaction of Fe(CN) 4-/3-

* e.u. = cal. m o l - i. deg- i.

TABLE lI OBSERVED RATE CONSTANTS FOR THE HYDROXYETHYL F E R R O C E N E R E D U C T I O N OF NATIVE A N D T2D LACCASE TYPE 1 Cu(lI) pH 7, 1 = 0.5 M (phosphate). [hydroxyethyl ferrocene] = 0.20 mM (T2D) or 0.91 mM (native). [laccase]=10 /~M. Second order rate constants calculated from k = kobJ[hydroxyethyl ferrocene] in activation parameter calculations. Uncertainty in kobs estimated at + 5%. Enzyme

Temperature (°C)

kob ~ (s -

Native laccase

7.2 8.3 11.1 14.4 15.3 16.1 20.1 21.8 24.2 26.4 26.7

0.144 0.246 0.300 0.695 0.841 1.10 1.20 1.80 2.75 4.95 5.12

T2 D laccase

7.1 12.7 17.0 28.1

0.170 0.458 1.02 5.91

1)

with type 2 copper evidently occurs in galactose oxidase [12]. Initial attack at or in the vicinity of type 2 Cu(II) appears to be a common feature in the oxidations of both organic [1,2,18] and inorganic [17] substrates by laccase blue copper. A remarkable range in both AH* (32 kcal/mol) and AS* (107 e.u.) is spanned within the family of blue copper protein reactions displayed in Table III. Considering laccase alone, increases in AH* and AS* of 15 kcal/mol and 57 e.u., respectively, occur along the series of Fe(II) reductants: Fe(EDTA) 2- , Fe(CN) 4-, Hydroxyethyl ferrocene. The activation enthalpy of 22.1 kcal/mol required to reduce T2D laccase type 1 Cu(II) with Fe(CN)64- is 13-16 kcal/mol larger than the analogous activation barriers of azurin and plastocyanin, consistent with previous findings on Fe(EDTA) 2- reductions [17]. Specific protein activation requirements clearly predominate over the Franck-Condon inner-sphere reorganization barrier in the electron transfer pathway to laccase type 1 copper. For this reason, the response of laccase reduction rate constants to variations in

115

thermodynamic driving force typically is not as expected from relative Marcus outer-sphere electron transfer theory [1,2,4]. Indeed, of the Fe(II)laccase reactions compared in Table III, hydroxyethyl ferrocene, a weak thermodynamic reductant ( E ° = +402 mV [19] compared to E ° (Fe(EDTA)-/2-)= +120 mV [20]), enjoys both the largest room temperature rate constant and, ironically, the most severe enthalpic activation barrier. In this context, a basis other than Marcus theory must be found to interpret quantitatively trends in activation parameters for blue copper protein redox reactions. The exceptionally large, positive activation entropies associated with the hydroxyethyl ferrocene and Fe(CN) 4- reductions of laccase blue copper imply extensive release of solvating water molecules and/or protein conformational movement preceding electron transfer [4,7,17]. The latter contribution certainly must be substantial, considering the consistently high A H* requirements in laccase reductions, even with a reagent susceptible to non-adiabatic electron transfer (Fe(EDTA) 2-) [21] and a physiological electron donor such as the hydroquinone monoanion (AH* = 10.7 kcal/mol,

AS* = 2 e.u.; I = 0.1 M) [17]. The compensation pattern between AH* and AS* in laccase reductions also is notable, as the activation parameters increase together within the series Fe(EDTA) 2-, Fe(CN)64-, hydroxyethyl ferrocene in such a way that only comparatively small differences in 298 K rate constants and AG* values exist. Indeed, an excellent linear relationship exists between AH* and AS*, with slope and correlation coefficient of 250 _+ 31 K and 0.993, respectively. Isokinetic relationships between A H* and AS* in the oxidations of cuprous plastocyanin and azurin by tris(1,10-phenanthroline)cobalt(II1) ions have shown that the mechanism is invariant with substitutions on the ligand aromatic ring system, in spite of substantial changes in activation parameters throughout the series [7]. Although the isokinetic relationship may be invoked with rigor only when structural variations within a series are modest [7,8], the compensation pattern in laccase reductions by Fe(EDTA) 2-, Fe(CN) 4- and hydroxyethyl ferrocene suggests a common mechanism, with considerable flexibility in activation requirements. Such flexibility in Fe(II) oxidations is most likely related to the hydrophilicity of the

TABLE Ill COMPARISON OF REDOX AGENTS

RATE

PARAMETERS

FOR

THE

REACTIONS

OF BLUE COPPER

PROTEINS

WITH

IRON(II,III)

C o n d i t i o n s of the p r e s e n t w o r k : p H 7, 1 = 0.5 M ( p h o s p h a t e ) . S t a n d a r d d e v i a t i o n s s h o w n in p a r e n t h e s e s . All a z u r i n entries p e r t a i n to the Pseudomonas aeruginosa p r o t e i n . H E F , h y d r o x y e t h y l f e r r o c e n e . Oxidant

Reductant

k ( 2 5 ° C ) ( M - 1. s - 1)

T2D laccase Fe(CN)~-

Fe(CN) 4T 2 D lacease

1,45 (0.03). 10 z 3,9 (0.3)-101

T2D laccase Native laccase Native laccase Stellacyanin Plastocyanin(bean) Azurin Fe(CN) 3-

HEF HEF Fe(EDTA) 2Fe(EDTA) 2Fe(EDTA) 2Fe(EDTA) 2plastocyanin (parsley) azurin Fe(CN)64Fe(CN) 4Fe(CN)64Fe(CN)64-

1,9.104 4,8-103 2.6.102 4.3.105 8.2.104 1.3.103

Fe(CN) 3Plastocyanin(parsley) Plastocyanin(spinach) Plastocyanin(bean) Azurin

a b c ¢

9.4.104 d 2.7.104 e 1.9.104 a 2 . 0 . 1 0 4 f'g 1.9.104 r 3.45-102 c

A H* (kcal/mol)

A S* (e.u.)

Ref.

22.1 (0.5)

+ 25 (2)

present work

28.0 28.0 13.0 3.0 2.2 2.0

(0.5) (1.4) b b c ¢

+ 55 (3) + 52 (5) - 5 b -- 21 b - 29 ~ - 37 c

present work present work 17 17 17 17

a e d r f c

-- 47 d - 52 * -- 17.5 - 9.1 - 10.6 - 27.1

11 13 11 unpublished data " unpublished data h 13

-- 3.3 -4.1 6.3 8.8 8.4 5.9

a f r *

a p H 6.9, 1 = 0.5 M. b p H 7.0, 1 = 0.1 M. c p H 7.0, 1 = 0.2 M. a p H 7.5, 1 = 0.10 M (NaCI). e p H 7.0, I = 0.22 M. f p H 6.0, I = 0.2 M (acetate). s 2 0 . 0 o c . h F e n s o m , D. a n d G r a y , H.B., c i t e d in Ref. 5.

116 electron donor, as AH*, AS* are smallest with the hydrophilic F e ( E D T A ) z - ion a n d most positive with hydroxyethylferrocene, m a d e slightly soluble in water only by virtue of its hydroxyl substituent. O n this basis, hydroxyethyl ferrocene should be the most efficient of the three ferrous complexes in p e n e t r a t i n g the h y d r o p h o b i c protein interior towards the solvent-inaccessible [4] type 1 site, while releasing its solvation sheath a n d p e r t u r b i n g the p r o t e i n c o n f o r m a t i o n to the greatest extent. Similar considerations evidently govern activation p a r a m e t e r s t h r o u g h o u t the entire family of blue copper protein redox reactions with Fe(II, III) reagents. Thus, a surprisingly good (correlation coeff i c i e n t = 0 . 9 9 0 ) linear correlation between A H * a n d AS*, with slope of 311 + 13 K, relates all of the activation parameters given in T a b l e III for F e ( E D T A ) 2 - , F e ( C N ) 4- , hydroxyethyl ferrocene reductions a n d F e ( C N ) 3- oxidations. W e t h a n k the R o b e r t A. Welch F o u n d a t i o n for s u p p o r t of this research through grants D-735 (R.A.H.) a n d D-710 (D.B.K.).

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