Reorganization Energy of the CuA Center in Purple Azurin: Impact of

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J. Phys. Chem. B 2007, 111, 6690-6694

Reorganization Energy of the CuA Center in Purple Azurin: Impact of the Mixed Valence-to-Trapped Valence State Transition† Ole Farver,*,‡ Hee Jung Hwang,§ Yi Lu,*,§ and Israel Pecht*,⊥ Institute of Analytical and Pharmaceutical Chemistry, UniVersity of Copenhagen, DK-2100 Copenhagen, Denmark, Department of Chemistry, UniVersity of Illinois at Urbana-Champaign, Urbana, Illinois 61801, and Department of Immunology, The Weizmann Institute of Science, RehoVot 76100, Israel ReceiVed: NoVember 3, 2006; In Final Form: December 24, 2006

Mixed valence (MV) coordination compounds play important roles in redox reactions in chemistry and biology. Details of the contribution of a mixed valence state to protein electron transfer (ET) reactivity such as reorganization energy, however, have not been experimentally defined. Herein we report measurements of reorganization energies of a binuclear CuA center engineered into Pseudomonas aeruginosa azurin that exhibits a reversible transition between a totally delocalized MV state at pH 8.0 and a trapped valence (TV) state at pH 4.0. The reorganization energy of a His120Ala variant of CuA azurin that displays a TV state at both the above pH values has also been determined. We found that the MV-to-TV state transition increases the reorganization energy by 0.18 eV, providing evidence that the MV state of the CuA center has lower reorganization energy than its TV counterpart. We have also shown that lowering the pH from 8.0 to 4.0 results in a similar (∼0.4 eV) decrease in reorganization energy for both blue (type 1) and purple (CuA) azurins, even though the reorganization energies of the two different copper centers are different at a given pH. These results suggest that the MV state plays only a secondary role in modulation of the ET reactivity via the reorganization energy, as compared to that of the driving force.

Introduction CuA is a binuclear copper center, with each copper ion coordinated by a histidine imidazole and both copper ions bridged by two cysteine thiolates (Figure 1).1-4 It serves as an electron transfer (ET) center in cytochrome c oxidase (CcO)5 and nitrous oxide reductase.6 The former is the terminal oxidase in the aerobic respiration chain, reducing dioxygen to water, and converting the redox energy into a proton gradient that provides a basis for the bioenergetic process leading to the synthesis of adenosine triphosphates (ATP) by ATP synthase.7-10 The CuA center is the initial electron uptake site of CcO from cytochrome c transferring it intramolecularly to the heme a center.11 Important questions are (1) what makes this ET so fast (kET >10 000 s-1),12,13 despite the relatively low driving force (10-90 mV); (2) how are the redox properties of CuA (a) fine-tuned; and (b) coupled to the proton gradient formation process? Answers to the above questions may lie in the regulation mechanism of the mixed valency of the CuA center. This center contains a single unpaired electron that is fully delocalized on the two copper sites.2-4,14-16 Until recently,17-24 such a discrete fully delocalized mixed valent (MV) copper center was rarely known in either chemistry or biology; most known binuclear copper centers contained trapped valence (TV) species in which the unpaired electron is either partially or fully residing on one of the two copper †

Part of the special issue “Norman Sutin Festschrift”. * Corresponding authors. (O.F.) Phone: +45 3530 6269. Fax : +45 3530 6013. E-mail: [email protected]. (I.P.) Phone: +972 8934 4020. Fax: +972 8946 52 64. E-mail: [email protected]. (Y.L.) Phone: +1 217 244 3186. Fax: +1 217 333 2685. E-mail: [email protected]. ‡ University of Copenhagen. § University of Illinois at Urbana-Champaign. ⊥ The Weizmann Institute of Science.

ions.25,26 Even less known was a reversible transition between the MV and TV states.27,28 Therefore, it was exciting to find out that the CuA center engineered into Pseudomonas aeruginosa azurin (called purple azurin) exhibits a MV state at high pH (>7) that can be converted into a TV state upon lowering the pH (to ∼4) in a completely reversible manner.29 Furthermore, site-directed mutagenesis had identified a C-terminal histidine (His120) as the site of this protonation, because mutation of His120 into Ala abolished the pH-dependent valence state transition.29-31 However, it should be added that although the nature of TV state of the H120 mutants has been debated,32 EPR measurements showed a four-line hyperfine characteristic of an electron localized in one of the two copper ions.16,30,31 Interestingly, this mutation resulted in a TV center with an increase in reduction potential (∼70 mV). Since His120 corresponds to the C-terminal histidine in CcO located along the ET pathway between CuA and its partner, heme a, the increase in reduction potential is sufficiently large to prevent ET from CuA to heme a. This finding yielded the idea that the proton pumping across the membrane may be controlled by redox properties of the CuA site (Figure 1C):29 At neutral pH, the C-terminal histidine is deprotonated, and the CuA center is in a MV state with a small (∼50 mV) lower reduction potential than its redox partner, heme a,33,34 resulting in ET flow that drives proton pumping across the membrane. However, when excess protons are accumulated in the periplasmic space, the C-terminal histidine, which is at the interface between the periplasmic space and the membrane, will become protonated. The protonation will promote transition to the TV state, raise the CuA reduction potential (>70 mV),29 prevent the ET flow, and thus stop proton pumping until those excess protons are used by ATP synthase.

10.1021/jp0672555 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/03/2007

Impact of MV-to-TV State Transition for CuA

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Figure 1. (A) The CuA center in purple azurin; (B) overlay of the two peptide patches connecting electron donor and acceptor in blue copper azurin (light blue) and purple CuA azurin (dark blue) from P. aeruginosa; (C) schematic representation of possible regulation of proton pumping through protonation of histidine and MV-to-TV transition.

Although the above study29 suggested a thermodynamic control of ET, the impact of an MV-to-TV transition on the kinetic parameters of the ET reactivity is unknown. Previous theoretical and spectroscopic studies have suggested that the fully delocalized MV state of CuA may be more efficient in ET than the TV state of the same center by lowering the reorganization energy.2,14,15,35 However, few experimental studies have addressed questions such as what effect, if any, the valence state and pH have on the ET-rate-determining parameters, such as the reorganization energy. To try to answer the above questions, we have earlier investigated the ET reactivity of the engineered CuA center in purple azurin using pulse radiolysis.36 Results of this study have shown a lower reorganization energy requirement for ET of CuA, as compared with that of the blue, type 1, Cu site, both located in the same protein frame, that is, in azurin.36 Since these results were obtained at pH 5.0, at which a considerable fraction of the CuA site has recently been shown to be in the TV state, we have now extended our studies of the intramolecular RSSR- to CuA ET in both purple azurin and its His120Ala mutant at higher and lower pH to determine the reorganization energy difference between MV and TV CuA.

Experimental Methods Sample Preparations. CuA azurin37,38 and its His120Ala variant30,31 were constructed, expressed in BLR(DE3) Escherichia coli (Novagen, Madison, WI), and purified as previously described. The DNA sequences were confirmed at the DNA sequencing facility of the University of Illinois Biotechnology Center, and the molecular weights of the proteins were verified by electronspray mass spectrometry at the University of Illinois Mass Spectrometry Center. As reported previously,29 the purple azurin at pH 7 displays strong visible absorption bands at 485, 530, and 770 nm. Upon lowering the pH to ∼4, the bands redshifted to 490, 530, and 810 nm, respectively, with an isosbestic point at 767 nm. EPR studies indicated that the lowering of the pH resulted in a transition from MV to TV. The mutant protein, His120Ala, on the other hand, displayed no change in UV-vis or EPR properties, suggesting the presence of a TV state in His120Ala azurin at all pH values.29 Kinetic Measurements. The pulse radiolysis experiments were carried out using the Varian V-7715 linear accelerator at the Hebrew University in Jerusalem. Accelerated electrons (5 MeV) at pulse lengths in the range from 0.1 to 1.5 µs produced 0.6-10 µM of CO2- radical ions under the present experimental

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Farver et al.

Figure 2. Time-resolved absorbance changes of a 15 µM purple azurin solution following pulse radiolysis in a N2O-saturated 100 mM formate solution, 10 mM phosphate at 10.8 °C, pH 4.0. The pulse width was 0.2 µs, and the light pass was 12.3 cm. The time scale is in seconds; the amplitudes are in cm-1. (A) monitored at 410 nm, reflecting formation and decay of the RSSR- radical; (B), monitored at 485 nm, showing reduction of the CuA center.

TABLE 1: Rate Constants, Activation, and Thermodynamic Parameters of Intramolecular ET at 298 K CuA azurin

kET, s-1 ∆H*, kJ mol-1 ∆S*, J K-1 mol-1 ∆G0, kJ mol-1 G0, eV λTOT, eV

H120A

pH ) 4.0 (TV)

pH ) 8.0 (MV)

pH ) 4.0 (TV)

pH ) 8.0 (TV)

981 ( 9 34.0 ( 4.1 -73 ( 6 -71.11 -0.737 0.75

104 ( 2 27.3 ( 5.4 -114 ( 19 -55.48 -0.575 1.07

873 ( 4 31.5 ( 4.1 -83 ( 6 -71.69 -0.743 0.84

41 ( 8 41.2 ( 8.2 -78 ( 17 -60.21 -0.624 1.25

conditions (cf. below). All optical measurements were carried out anaerobically under purified N2O in either a 1 cm Suprasil cuvette (Hellma) or a 4 cm Spectrosil cuvette using three light passes, which results in an overall optical path length of 12.3 cm. A 150 W xenon lamp produced the analyzing light beam, and to avoid photochemistry and light scattering, an optical filter with cutoff at 385 nm was used. The data acquisition system consisted of a Tektronix 390 A/D digitizer connected to a PC. The temperature range employed in the kinetic studies, 273313 K, was controlled by a thermostat and continuously monitored by a thermocouple attached to the cuvette. The protein concentration was varied between 15 and 150 µM. All reactions were performed under pseudo-first-order conditions, with typically a 10-fold excess of oxidized protein over reductant. The concentration of oxidized CuA was monitored at 485 nm (485 ) 3500 M-1 cm-1), whereas formation and decay of the RSSRradical were followed at 410 nm (410 ) 10,000 M-1 cm-1). Each individual kinetic measurement has been repeated at least three times at each temperature. The data were analyzed by fitting to a sum of exponentials using a nonlinear least-squares program written in MATLAB (The MathWorks, Natick, MA). Aqueous solutions, 0.1 M in sodium formate, 10 mM phosphate were deaerated and saturated with N2O by bubbling directly in the cuvette. The pH was kept constant at either 4.0 or 8.0. Afterward, the concentrated protein stock solution was added, and N2O bubbling was continued for another 5 min before pulsing the protein solution. Results and Discussion Pulse radiolytically produced CO2- radicals reduce the disulfide bridge as well as the CuA site of purple azurin and its His120Ala mutant. The radical concentration was always sufficiently low so that less than 2% of the protein’s electron acceptor sites were reduced following a single pulse. Hence, the probability for any azurin molecule being reduced by more than one electron equivalent following one pulse is low.

Processes in the microsecond time range were monitored at 410 nm, where the RSSR- radical anion absorbs (Figure 2A, left panel), and also at 485 nm, where the oxidized CuA center absorbs (Figure 2B, left panel). Both of these reduction processes were found to be direct, second-order reactions with the CO2radicals. These fast, diffusion-controlled bimolecular reactions were followed by slower processes in the millisecond time range that were earlier shown to reflect the intramolecular ET from the disulfide radical ion (Figure 1A, right panel) to the oxidized CuA center (Figure 1B, right panel). The observed rate constants of CuA reduction and RSSR- reoxidation at a given pH value were found to be identical within experimental error and independent of both protein and CO2- radical concentrations, demonstrating that an intramolecular process takes place:

kET Az[CuA(ox)RSSR ] f Az[CuA(red)RSSR] -

The temperature dependence of this process was further examined for both CuA azurin and its His120Ala mutant in a 0-40 °C range at the two pH values. The observed rate constants and calculated driving forces of the ET reactions at pH 4.0 and 8.0, as well as the derived activation parameters, are shown in Table 1. According to the semiclassical Marcus theory39 for ET between reactants in spatially fixed and oriented sites, the firstorder rate constant can be expressed as a product of an electronic and an exponential, nuclear factor,

{

kET ) κ(r)ν exp -

}

∆G * RT

(1)

where κ(r) is the electronic transmission coefficient when the reactants are at a distance, r, apart, and ν is the nuclear frequency factor (1 × 1013 s-1).39

Impact of MV-to-TV State Transition for CuA

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In the nonadiabatic regime where κ(r) , 1, κ(r)ν becomes independent of the frequency of nuclear motion, and

κ(r)ν )

HDA2

2π p (4πλ

1/2

TOTRT)

{

λTOT ∆G0 1+ 4 λTOT

}

-r

H120A H120A - λSS(4) ) ) 1.25 - 0.84 eV ) 0.41 eV + 1/2 (λSS(8)

We have earlier determined the rate constants of the intramolecular ET in wild type, type 1 copper (WT T1Cu) azurin in the pH range 4-8, where kET varies from 285 to 15 s-1 at 298 K.41 From the difference in driving force (0.066 eV), we calculated the difference in reorganization energy due to the pH change to be

2

WT T1Cu: λTOT(blue, 8) - λTOT(blue, 4) ) 0.42 eV

(3)

while as shown above in H120A,

When the driving force of the reaction equals the total reorganization energy, ∆G* becomes 0, and the rate constant reaches its maximum value, kMAX ) κ(r)ν. HDA2 decays exponentially with the separation distance, and we can write κ(r)ν as39

κ(r)ν ) 1 × 1013 e{-β(r

H120A H120A H120A H120A λTOT(TV, 8) - λTOT(TV, 4) ) 1/2 (λCu(TV, 8) - λCu(TV, 4))

(2)

HDA is the matrix element describing the coupling of donor and acceptor electronic states.39 The activation free energy can be expressed in terms of driving force (-∆G0) and reorganization energy, λTOT.

∆G * )

1. The pH Effect on λ (Using H120A Data).

s-1

∆λTOT ) 0.41 eV 2. The Valence State Effect on λ. CuA H120A CuA H120A λTOT(MV, 8) - λTOT(TV, 8) ) 1/2 (λCu(MV, 8) - λCu(TV, 8))

(4)

CuA H120A - λSS(8) ) + 1/2 (λSS(8)

where r0 is the value of r for D and A in direct (van der Waals) contact while the generally accepted value for r0 is 3.0 Å. A time table for activationless electron tunneling in β-sheet proteins predicts a coupling decay constant, β ) 1.0 Å-1.40 Using the closest distance between the binuclear CuA center and the Cys3/26 disulfide group of 26.04 Å42 and applying eq 4, we find kMAX ) 986 s-1, that is, a rate constant for an essentially activationless ET process in CuA azurin at pH 4.0, indicating that λTOT must be close to the value for the driving force, -∆G0. Since

Since little change in the disulfide bonds between purple azurin and its H120A mutant is expected, we can assume

ln kMAX ) ln kET +

0)}

∆G * RT

(5)

we can calculate the total reorganization energy from the known kET and driving force using eqs 5 and 3 above. Results of these calculations are presented in Table 1. According to the Marcus cross-relation, the total reorganization energy, λTOT is related to the reorganization energies of the individual redox centers approximately as39

λCu λSS + λTOT ) 2 2

CuA H120A λSS(8) ) λSS(8)

Then CuA H120A CuA H120A λCu(MV, 8) - λCu(TV, 8) ) 2[λTOT(MV, 8) - λTOT(TV, 8)] )

2[1.07 - 1.25] ) -0.36 eV This number includes both the difference between MV and TV and the difference of metal sites due to the mutation. Since at pH 4, both purple azurin and its H120A mutant are in the TV state, the difference in metal sites due to mutation can be obtained from CuA H120A CuA H120A λTOT(TV, 4) - λTOT(TV, 4) ) 1/2 (λCu(TV, 4) - λCu(TV, 4)) CuA H120A - λSS(4) ) + 1/2 (λSS(4)

Again, assuming CuA H120A ) λSS(4) λSS(4)

(6)

where Cu refers to the CuA center, and SS, to the disulfide group. Below, numbers 4 and 8 refer to pH. The other symbols have already been defined above. For purple CuA azurin, CuA CuA CuA λTOT(MV, 8) ) 1/2 (λCu(MV, 8) + λSS(8)) CuA CuA CuA λTOT(TV, 4) ) 1/2 (λCu(TV, 4) + λSS(4))

For the H120A variant of purple CuA azurin, H120A H120A H120A λTOT(TV, 8) ) 1/2 (λCu(TV, 8) + λSS(8) ) H120A H120A H120A λTOT(TV, 4) ) 1/2 (λCu(TV, 4) + λSS(4) )

We can now perform the following calculations, using the results presented in Table 1:

then CuA H120A CuA H120A λCu(TV, 4) - λCu(TV, 4) ) 2[λTOT(TV, 4) - λTOT(TV, 4)] )

2[0.75 - 0.84] ) -0.18 eV Therefore, the difference for the MV-to-TV transition becomes

-0.36 eV - (-0.18 eV) ) -0.18 eV A very interesting finding of this study is that the pHdependent change in reorganization energy is essentially identical whether CuA or a blue, T1, copper is residing in azurin. In other words, even though the absolute reorganization energy values of the two copper centers are different, their changes with pH are the same. Comparison of the 3-D structures of purple and WT azurins indicates that, with the exception of the loop region where the respective copper centers reside, the overall structures of the two proteins are the same, and one of the copper ions in the binuclear CuA center also overlays well the mononuclear blue T1 copper (Figure 1B).42 The fact that

6694 J. Phys. Chem. B, Vol. 111, No. 24, 2007 both copper centers are in an almost identical environment may be the reason for the similar pH-dependent increase of reorganization energy. It further demonstrates that protein design that places distinct metal centers into the same protein framework is an effective approach that can minimize the protein matrix effect. Despite this fact, it is still remarkable to observe that, within the same environment, the change in reorganization energies of the two ET centers are the same, even though they have very different structures. The Isolated Spin Change Effect. Upon transition from the trapped valence (TV) to the mixed valence (MV) state, λTOT decreases by 0.09 eV. This is a very small decrease, amounting to only 20% of the pH effect on reorganization energies. Assuming (safely) that the disulfide sites in purple and H120A azurin have identical reorganization energies, we calculated the spin change effect on the CuA center above: λCu will decrease by 0.18 eV upon the TV to MV transition. Conclusions The influence of the valence state of the binuclear CuA center on the kinetic ET parameters has been examined using an engineered purple CuA center and its variant at two different pH conditions. We have obtained direct experimental evidence that the MV state of the CuA center has a lower reorganization energy than its TV counterpart, yet the magnitude of the decrease (0.18 eV) is surprisingly small in comparison with the marked change of reorganization energy caused by the pH change (0.4 eV). An earlier report has indicated that the MVto-TV transition results in an ∼70 mV increase in reduction potential, enough to reverse the ET flow from heme a to CuA in CcO and, thus, could shut down the proton pumping.29 Therefore, the results presented here suggest that, in comparison to the earlier reported thermodynamic control through modulation of reduction potentials (i.e., driving force),29 kinetic control by modulation of the reorganization energy appears to be much more subtle and only plays a secondary role. However, in light of the large number of different factors influencing the reorganization energy, our determination of the electronic contribution to λ is one step toward a full understanding of this parameter. Acknowledgment. This material is based upon work supported in part by the U.S. National Science Foundation (CHE05-52008 to Y.L.). The support extended to I.P. by the Minerva Foundation, Munich, Germany is gratefully acknowledged. We thank Dr. Ulf Ryde, Department of Theoretical Chemistry, Lund University, for helpful discussions. References and Notes (1) Beinert, H. Eur. J. Biochem. 1997, 245, 521. (2) Randall, D. W.; Gamelin, D. R.; LaCroix, L. B.; Solomon, E. I. J. Biol. Inorg. Chem. 2000, 5, 16. (3) Vila, A. J.; Ferna´ndez, C. O. Copper in Electron Transfer Proteins.In Handbook on Metalloproteins; Bertini, I., Sigel, A., Sigel, H., Eds.; Marcel Dekker: New York, 2001; pp 813. (4) Lu, Y. Electron transfer: Cupredoxins. In ComprehensiVe Coordination Chemistry II: From Biology to Nanotechnology; McCleverty, J. A., Meyer, T. J. Eds.; Elsevier: Oxford, UK, 2004; Vol. 8 (Biocoordination Chemistry; Que, J., Lawrence, Tolman, W. B., Eds.), pp 91.

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