Entropic Parameters of a Protein Redox Center

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Tuning of Enthalpic/Entropic Parameters of a Protein Redox Center through Manipulation of the Electronic Partition Function Damian Alvarez-Paggi,†,§ Ulises A. Zitare,† Jonathan Szuster,† Marcos N. Morgada,‡ Alcides J. Leguto,‡ Alejandro J. Vila,‡ and Daniel H. Murgida*,† †

Instituto de Química Física de los Materiales, Medio Ambiente y Energía (INQUIMAE), Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and CONICET,1428 Buenos Aires, Argentina ‡ Instituto de Biología Molecular y Celular de Rosario (IBR), Departamento de Química Biológica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario and CONICET, 2000 Rosario, Argentina S Supporting Information *

ABSTRACT: Manipulation of the partition function (Q) of the redox center CuA from cytochrome c oxidase is attained by tuning the accessibility of a low lying alternative electronic ground state and by perturbation of the electrostatic potential through point mutations, loop engineering and pH variation. We report clear correlations of the entropic and enthalpic contributions to redox potentials with Q and with the identity and hydrophobicity of the weak axial ligand, respectively. Figure 1. Left: Crystallographic structure of the Tt-CuA domain (pdb 2CUA). The loops mutated in the Tt-3L chimera are indicated in red. Right: structural details of the CuA center.

P

roteins are dynamical entities that explore many conformations, which contribute to the partition function (Q).1−3 Hence, the large ensemble of states probed by proteins hampers the analysis of thermodynamics based on static structures. For redox metalloproteins, however, enthalpy contributions to redox potentials (E0) have been rationalized in terms of a static description, i.e., considering the factors that contribute to charge (de)stabilization such as the surrounding dielectric constant, specific electrostatic interactions and the nature of first and second sphere ligands.4−6 Entropy contributions are harder to analyze, as they report on the availability, multiplicity and population of the different energy levels available in each redox state. Factors that have been considered ad hoc to analyze redox entropy include solvent accessibility of the metal center, the chemical nature of ligands, redox state-dependent alteration of protein dynamics, changes in the coordination geometry of the metal center and noncovalent interactions.7−11 A robust theoretical and comprehensive model for entropy effects, however, remains elusive. For the most part, free energy surfaces are assumed “static”, arising from a single electronic state in the context of the Born−Oppenheimer approximation. However, quantum dynamical effects have been shown to affect systems such as photosynthetic reaction centers.12 The CuA site (Figure 1), the primary electron acceptor of cytochrome c oxidase (CcO),13 constitutes a bona f ide system to assess the effect on redox thermodynamics of manipulating the free energy landscape. As a type III mixed-valence (MV) species, formal charges in the oxidized and reduced forms are Cu1.5+Cu1.5+ and Cu1+Cu1+, respectively. In contrast to other biological metal centers, oxidized CuA can be described by a © 2017 American Chemical Society

double-well adiabatic potential energy surface as a function of small geometric distortions. 15−17 The lower lying well corresponds to a σu* ground state (GS), whereas the other one is a thermally accessible πu alternative GS. Recently, we showed that the σ*u /πu energy gap (ΔEσu*/πu) in Thermus thermophilus CuA (Tt-CuA; Figure 1)18 can be fine-tuned either by mutating the weak axial ligand M160, by acidification or by substitution of three loops flanking the metal center without altering the ligands (Tt-3L-CuA variant).19,20 Here, we untangle enthalpy/entropy contributions to E0 for CuA variants in which we have been able to manipulate the ΔEσu*/πu value and the electrostatic potential at the redox site. Dissection of the entropic component allows it to be described in terms of variations of Q and analyzing its dependence on ΔEσu*/πu. Enthalpic contributions are most likely ascribable to charge (de)stabilization. These effects were unraveled by studying different protein variants: (i) the wild type center Tt-CuA, (ii) first coordination sphere mutants M160Q and M160H, (iii) the chimeric protein Tt-3L and (iv) a variant with the M160H mutation in the Tt-3L scaffold (Figure 1). These species were spectroscopically characterized before, showing that the structural features and the MV character of the CuA site remain largely preserved.14,15,21 Cyclic voltammetry Received: May 19, 2017 Published: June 29, 2017 9803

DOI: 10.1021/jacs.7b05199 J. Am. Chem. Soc. 2017, 139, 9803−9806

Communication

Journal of the American Chemical Society Table 1. Redox Parameters and Energy Gaps Obtained for the Different CuA Variants at Two pH Values variant

a 0 EpH 3.5

(mV)

ΔEσu*/πupH 6.0 (cm−1)

261 ± 5 79 ± 5 93 ± 5 242 ± 5 74 ± 5

273 ± 5 110 ± 5 101 ± 5 268 ± 5 132 ± 5

600 ± 50 (5%) 200 ± 50 (28%) 900 ± 50 (1%) 240 ± 50 (24%) 12 ± 50 (49%)

(mV)

Tt-CuA M160H M160Q Tt-3L Tt-3L-M160H a

a 0 EpH 7.0

b

ΔEσu*/πupH 3.5 (cm−1)

ΔS0pH 7.0 (J K−1mol−1)

ΔS0pH 3.5 (J K−1 mol−1)

ΔH0pH 7.0 (kJ mol−1)

ΔH0pH 3.5 (kJ mol−1)

649 ± 70 (4%) 312 ± 70 (23%) 900 ± 70 (1%) 240 ± 70 (24%) 216 ± 70 (27%)

−42 ± 5 −45 ± 4 −7 ± 6 −39 ± 5 −47 ± 11

−27 ± 4 −12 ± 11 5±5 −35 ± 8 −5 ± 8

−38 ± 2 −21 ± 1 −11 ± 1 −35 ± 2 −21 ± 2

−34 ± 1 −14 ± 3 −8 ± 1 −36 ± 2 −14 ± 2

c

Values vs NHE. bDetermined by NMR.14,15 cDetermined by UV−vis. Numbers in parentheses are populations of the πu GS at 298 K.

phobicity of the axial ligand 160: −1.3, −0.5 and +0.2 for Met, His and Gln, respectively.22 This relationship can be ascribed to destabilization of Cu1.5+−Cu1.5+ relative to Cu1+−Cu1+ upon increasing the hydrophobicity of the metal environment. Note that although blue copper sites exhibit good correlations of E0 with the hydrophobicity of the axial ligand,23−25 there is no clear tendency for the E0 values of CuA sites (Table 1). This observation reinforces the idea that charge (de)stabilization in CuA mainly affects the energetic term, and hints at a special role of the entropic contribution to E0. Combining eqs 2 and 3, ΔS can be expressed as

(CV) determinations, however, reveal a 200 mV modulation of E0 (Figure S1 and Table 1). The Helmholtz energy change of reduction (which can be approximated to ΔG) arises from the energetic and entropic contributions and is related to Q:

A = −kBT lnQ

(1)

ΔA = ΔU − T ΔS

(2)

⎛Q ⎞ ΔA = −kBT ln⎜⎜ Red ⎟⎟ ⎝ Q Ox ⎠

(3)

⎛ Q ⎞ ΔU ΔS = kB ln⎜⎜ Red ⎟⎟ + T ⎝ Q Ox ⎠

0

To determine the entropy/enthalpy contributions to E , CVs of the different protein variants in solution were recorded as a function of temperature (ca. 5−40 °C), both at neutral and acidic pH. Representative CVs and variations of E0 with T are shown in Figures S2−S4. The enthalpic component is extracted from the temperature dependence of E0: ⎛∂ −ΔH ° ⎜ =⎜ nF ⎝∂

( TE ) ⎞⎟ ( T1 ) ⎟⎠p

(5)

The partition function is usually separated into electronic, vibrational, rotational and conformational contributions:

(4)

ΔH0 values determined by this procedure are compiled in Table 1. Note that for the series Tt-CuA/M160H/M160Q ΔH0 varies ca. 20 kJ mol−1. In contrast, the variants Tt-CuA and Tt-3L, which share the same set of first ligands but differ in the sequences of the surrounding loops, exhibit very similar ΔH0 values. A similar scenario is verified for M160H and Tt-3LM160H, thus underlining that ΔH0 is mostly determined by the chemical nature of first sphere ligands (specifically at position 160) but not by the surrounding environment. Moreover, ΔH0 is largely insensitive to pH for all the studied proteins. As shown in Figure 2, ΔH0 exhibits a good correlation with the hydro-

Q = Q ElQ VibQ RotQ Conf

(6)

⎛Q ⎞ Q Q Q Red,El Red,Vib Red,Rot Red,Conf ⎟ + ΔU ΔS = kB ln⎜⎜ ⎟ T ⎝ Q Ox,ElQ Ox,VibQ Ox,RotQ Ox,Conf ⎠

(7)

Although such approximation may not strictly apply for CuA sites due to conformationally coupled σ*u /πu interconversion, it represents a useful first-order approach to evaluate the effect of QEl on ΔS. Moreover, the comparison among protein variants that are structurally very similar14,15,21 is expected to average out the coupling effect, at least partially. From an electronic point of view, the MV oxidized form is a doublet for both σu* and πu alternative GS, whereas the reduced state is a singlet and presents a single-well potential energy surface.16,17 Therefore, the electronic degeneracy is 1 for the reduced state and 2 for each of the GS in the oxidized form. The electronic energies of the MV πu GS and of the reduced state can be expressed relative to the MV σu* GS employing ΔEσu*/πu and ΔU, respectively. Taking into account these considerations, we can express: ⎛ ′ ⎞ ΔU Q Red e−ΔU / kBT ⎟+ ΔS = kB ln⎜⎜ −ΔE /k T ′ ⎟⎠ T ⎝ 2 + 2e σu*/πu B Q Ox

(8)

⎛Q′ ⎞ ΔS = −kB ln(2 + 2e−ΔEσu*/πu / kBT ) + kB ln⎜⎜ Red ⎟⎟ ′ ⎠ ⎝ Q Ox

(9)

where QRed ′ and QOx ′ are the nonelectronic contributions to Q in the reduced and oxidized states, respectively, i.e., ΔS = ΔSEl + ΔS′. To verify this ΔS/ΔEσu*/πu relationship, ΔS0 values were determined from temperature-dependent CVs (Figure S3)

Figure 2. Experimental ΔH0 values of reduction as a function of the hydrophobicity of the axial ligand. Solid circles: values at pH 7.0. Hollow circles: values at pH 3.5. 9804

DOI: 10.1021/jacs.7b05199 J. Am. Chem. Soc. 2017, 139, 9803−9806

Communication

Journal of the American Chemical Society

Interestingly, the NMR signals of the Tt-CuA, M160H and Tt3L-M160H variants exhibit pH-dependence qualitatively similar to the effect of lowering the temperature at fixed pH (Figures S5−S9). This is indicative of a higher ΔEσ*u /πu value at low pH, which however could not be determined by NMR due to protein instability in acidic media and the long acquisition times of the NMR experiments. Therefore, these values were estimated from the UV−vis spectra (see below). Most likely the observed pH dependence arises from an increase in solvent accessibility at higher pH, as suggested before for WT Tt-CuA.21 The variants M160Q and Tt-3L are insensitive to pH, at least within the explored range (2 to 7). We have previously reported the variation of ΔEσu*/πu with pH for M160H.19 To analyze the variation of ΔS with ΔEσu*/πu, we estimated here this pHdependence for all the studied proteins. To this end, temperature-dependencies of the UV−vis absorption spectra were determined at pH 7.0 and 3.5 (Figure S10). Relative σ*u /πu populations were estimated as described in the SI from the intensity ratio of the SCys→Cu charge transfer bands, which for the σ*u and πu GS appear at 540 and 357 nm, respectively (Table 1).15 Note that there is no clear correlation between E0 and ΔEσu*/πu, but all protein variants experience 20−50 mV upshifts of E0 upon lowering the pH. Thus, some of the introduced structural modifications and variation of pH tune the values of both E0 and ΔEσu*/πu. At acidic pH the variants M160Q, Tt-CuA and Tt-3L also exhibit the predicted variation of ΔS0 with ΔEσu*/πu, whereas M160H and Tt-3L-M160H deviate from this general trend. This is not surprising as previous studies show that replacement of the weak axial ligand M160 by His does not disrupt the geometry nor the MV character of the site, but renders the kinetic ET parameters sensitive to pH.15 Notably, Tt-3L is resilient to pH variations even though ΔEσ*u /πu is small and comparable to that of M160H. Thus, we conclude that the deviations of the M160H and Tt-3L-M160H variants at low pH reflect nonelectronic contributions to Q that arise from subtle pH-dependent conformational changes. In agreement with this interpretation, the difference of reaction entropies (neutral minus acidic, ΔΔS0), which is expected to cancel out largely these nonelectronic contributions, exhibits a clear dependence with ΔΔEσ*u /πu (Figure 3B). Moreover, there is a clear correlation between electrochemically obtained ΔΔS0 values (ΔΔS0exp) and those calculated for the electronic part (ΔΔSCalc El ) employing the first term of eq 9 and ΔEσ*u /πu values from Table 1 (Figure 3C; see SI for details). The fact that the experimental values exceed the calculated electronic contributions suggests that Q′Red and Q′Ox are also affected by ΔEσu*/πu and are not independent of QEl. Attempts to asses Q′Red and Q′Ox by calorimetry were unsuccessful as none of the studied proteins displayed a clear melting below 100 °C.26 Although the variation of E0 ultimately arises from differential ligand field effects among protein variants, the statistical model assumed here predicts that only ΔS0 but not ΔH0 should be affected by the multiplicity of the different redox states, as verified experimentally. This report constitutes, to the best of our knowledge, the first semiquantitative rationalization of reduction potentials of biological redox centers in terms of the partition function, which is modulated through the thermal accessibility of a low lying alternative GS. The impact of this effect on ΔS0 can account for variations of E0 up to 90 mV (e.g., M160Q vs

employing eq 10, where n, F and T have their usual meanings. The obtained ΔS0 values are summarized in Table 1. ⎛ ∂E 0 ⎞ ΔS ° = nF ⎜ ⎟ ⎝ ∂T ⎠ p

(10)

For the oxidized proteins at pH 6.0 ΔEσu*/πu was determined before by temperature-dependent NMR (Table 1). Given that the UV−vis absorption spectra at pH 6 and 7 are identical, we can confidently assume that ΔEσu*/πu values are also similar (see below). According to the statistical model represented by eq 8, ΔSEl should be negative, as it involves a transition from a state of degeneracy between 2 and 4, depending on the ΔEσu*/πu/RT ratio, to a nondegenerate state. Thus, if all other contributions remain constant, the absolute value of ΔS0 should increase when ΔEσu*/πu decreases. As shown in Figure 3A, the experimental results at neutral pH exhibit the predicted trend. For Tt-3LM160H, where both GS are almost equally populated at room temperature, the modulus of ΔS0 is about seven times larger than that of M160Q, which presents the least accessible πu state.

Figure 3. (A) Variation of ΔS0 with ΔEσu*/πu at pH 7.0. (B) ΔΔS0 as a function of ΔΔEσu*/πu. The double difference refers to values at neutral minus acidic pH. (C) Correlation between ΔΔSCalc calculated as El indicated in the main text and ΔΔS0 obtained electrochemically. 9805

DOI: 10.1021/jacs.7b05199 J. Am. Chem. Soc. 2017, 139, 9803−9806

Journal of the American Chemical Society

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M160H), which represents a large shift of the driving force and, therefore, may affect the kinetics of inter- and intraprotein ET. Moreover, the pH dependence of ΔEσu*/πu suggests new ways in which local pH could play a role in controlling protein ET, particularly in respiratory and photosynthetic redox chains. The present results may reconcile previous contradictory reports on the role of the weak axial ligand on E0,27,28 as dissection of enthalpy/entropy contributions show the markedly different effects of each ligand. Although most metal sites show variability in the ligand composition, CuA remains unchanged across proteins and species. Our present and recent findings may help explain this invariability.15,19−21 Nature preserves an axial ligand that (i) coordinates the site conferring structural rigidity to perform a reversible redox reaction (ii) impacts on the hydrophobicity affecting E0 through ΔH0 (iii) fine-tunes the accessibility of the alternative GS (iv) regulates the reorganization energy (λ) of the site, (v) modulates the covalencies in each GS thus affecting the electronic coupling and (vi) modulates E0 through the variation of ΔS0. All these parameters, in turn, affect ET kinetics.29,30 Moreover, recent theoretical calculations15 showed that ΔEσu*/πu may be susceptible to subtle geometric distortions of the CuA site that might be brought about by protein−protein interactions. Biologically relevant electric fields have also been shown to modulate ΔEσu*/πu.15 In light of the present results, these observations hint at a novel mechanism for fine-tuning the thermodynamic and kinetic parameters of the ET reaction.31 For the specific case of CcO, it appears that nature has evolved the specific constrains and pH sensitivity mentioned above to optimize the CuA site in terms of ET driving force, λ and ability to redirect incoming and outgoing electrons. Tuning of Q through ΔEσu*/πu seems to be one of the pathways of evolutionary optimization. The ubiquitous nature of CuA, present in NO- and N2O-reductases and in most O2-reductases throughout the three domains of life, underlines the relevance on these findings. Moreover, similar entropic control of E° might apply to other metal sites, such as in blue copper and heme proteins, which may thermally populate distorted configurations.32,33

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b05199. NMR and UV−vis spectra, electrochemical data, experimental details (PDF)

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Communication

AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Daniel H. Murgida: 0000-0001-5173-0183 Present Address

́ Instituto de Investigaciones Bioquimicas de Buenos Aires, FILCONICET. §

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank ANPCyT and UBACYT for financial support and Prof. G. Montich for DSC determinations. 9806

DOI: 10.1021/jacs.7b05199 J. Am. Chem. Soc. 2017, 139, 9803−9806