Discrete Ligand Binding and Electron Transfer Properties of

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Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

Discrete Ligand Binding and Electron Transfer Properties of ba3‑Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments Constantinos Koutsoupakis,† Tewfik Soulimane,‡ and Constantinos Varotsis*,† †

Department of Environmental Science and Technology, Cyprus University of Technology, P.O. Box 50329, 3603 Lemesos, Cyprus Chemical and Environmental Science Department and Materials & Surface Science Institute (MSSI), University of Limerick, Limerick, Ireland

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‡

CONSPECTUS: Cytochrome c oxidase (CcO) couples the oxidation of cytochrome c to the reduction of molecular oxygen to water and links these electron transfers to proton translocation. The redox-driven CcO conserves part of the released free energy generating a proton motive force that leads to the synthesis of the main biological energy source ATP. Cytochrome ba3 oxidase is a Btype oxidase from the extremely thermophilic eubacterium Thermus thermophilus with high O2 affinity, expressed under elevated temperatures and limited oxygen supply and possessing discrete structural, ligand binding, and electron transfer properties. The origin and the cause of the peculiar, as compared to other CcOs, thermodynamic and kinetic properties remain unknown. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR (TRS2-FTIR) spectroscopies have been employed to investigate the origin of the binding and electron transfer properties of cytochrome ba3 oxidase in both the fully reduced (FR) and mixed valence (MV) forms. Several independent and not easily separated factors leading to increased thermostability and high O2 affinity have been determined. These include (i) the increased hydrophobicity of the active center, (ii) the existence of a ligand input channel, (iii) the high affinity of CuB for exogenous ligands, (iv) the optimized electron transfer (ET) pathways, (v) the effective proton-input channel and water-exit pathway as well the proton-loading/exit sites, (vi) the specifically engineered protein structure, and (vii) the subtle thermodynamic and kinetic regulation. We correlate the unique ligand binding and electron transfer properties of cytochrome ba3 oxidase with the existence of an adaption mechanism which is necessary for efficient function. These results suggest that a cascade of structural factors have been optimized by evolution, through protein architecture, to ensure the conversion of cytochrome ba3 oxidase into a high O2affinity enzyme that functions effectively in its extreme native environment. The present results show that ba3-cytochrome c oxidase uses a unique structural pattern of energy conversion that has taken into account all the extreme environmental factors that affect the function of the enzyme and is assembled in such a way that its exclusive functions are secured. Based on the available data of CcOs, we propose possible factors including the rigidity and nonpolar hydrophobic interactions that contribute to the behavior observed in cytochrome ba3 oxidase.

1. INTRODUCTION Aerobic respiration is one of the most exergonic metabolic processes known in nature.1,2 The free energy made available in exergonic redox chemistry is used to generate a proton electrochemical gradient required by ATP synthase to drive the synthesis of adenosine triphosphate (ATP). This proton motive force is achieved through two distinct mechanisms: charge separation and proton pumping.1−3 In charge separation, performed by both the cytochrome bd and hemecopper oxidoreductases, electrons originating from the periplasm and protons from the cytoplasm, catalyze the fourelectron reduction of molecular oxygen (O2) to water (H2O), assuring that the performed chemistry is coupled to charge separation across the membrane.1−3 In proton pumping, performed only by the heme-copper oxidoreductases, protons are taken from the cytoplasm, translocated across the membrane and released into the periplasm.1−6 The stoichiometric coefficient (n) of proton pumping is a measure of the efficiency of energy conservation and represents the number of © XXXX American Chemical Society

protons pumped per electron consumed during the oxidoreduction. The overall chemical reaction catalyzed by cytochrome c oxidases can be written as: O2 + 4cyt c(Fe 2 +) + 4H+in + 4nH+in ⇒ 2H 2O + 4cyt c(Fe3 +) + 4nH+out

where in and out refer to the internal space and the periplasmic or intermembrane space of the bacterial cell or the mitochondrion, respectively; n represents the quotient H+pump/e−.1−3 Three major oxygen reductase types, namely A, B, and C, have been identified based on genomic and structural analyses in the superfamily of the heme-copper oxidases.1−3 The superfamily is defined by the presence of a unique bimetallic active site, consisting of a high-spin heme, denoted a3, o3, or b3, Received: January 28, 2019

A

DOI: 10.1021/acs.accounts.9b00052 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research and a closely associated CuB in subunit I. Subunit I contains an additional low-spin heme a or heme b.1−6 Cytochrome ba3 oxidase from the thermophilic eubacterium Thermus thermophilus is a B-type oxidase,4 with high oxygen affinity expressed in the T = 47−85 °C range with optimum T = 70 °C and limited oxygen supply. As a bacterial terminal respiratory protein complex, it is located in the plasma membrane and catalyzes the four-electron reduction of molecular oxygen (O2) to water (H2O) with a simultaneously generation of an electrochemical proton gradient across the membrane.4−6 The 0.5 H+/electron pumping stoichiometry is lower than that of the 1 H+/electron found in other mitochondrial-like oxidases characterized to date.5 This gradient is created by the uptake of protons required for the formation of water and to a vectorial translocation of protons from the cytoplasmic to the periplasmic side of the membrane. In addition to the 4 e− reduction of O2, cytochrome ba3 also catalyzes the 2 e− reduction of nitric oxide (NO) to nitrous oxide (N2O),6−8 and the 2 e− oxidation of carbon monoxide (CO) to carbon dioxide (CO2).9,10 The determinant factor in the reactions of NO and CO is the binding constant (K) of CuB which acts as a ligand trap that controls the NO and CO activities.6−10 Cytochrome ba3 oxidase from T. thermophilus, shown in Figure 1, is the only B-type oxidase whose crystallographic structure

Figure 2. Structure of critical components of Thermus thermophilus cytochrome ba3 oxidase (PDB accession code 3S8G). (A) (CuA− CuA)-heme b-heme a3-CuB electron transfer (ET) pathway through Arg449, Arg450, His 386, Phe385, and His384 residues. The figure also includes the residues ligated to the five metal atoms, except His72 of heme b Fe atom. Two blue arrows (O2 channel, ET pathway) have been inserted to demonstrate the different relative orientation between Figures 1 and 2A. (B) K proton pathway of cytochrome ba3, used for the transfer of both chemical and pumped protons. The crystallographically characterized water molecules are shown as red spheres. This figure has been rotated 90° relative to the “K Proton Pathway” blue arrow of Figure 1. (C) Proton loading site (water pool), located in the moiety of the heme a3 propionates. The structurally resolved water molecules are shown as magenta spheres, and the residues surrounding them as sticks. This site is part (start) of the exit pathway for the catalytically produced H2O molecules and pumped protons. The hydroxyethylgeranylgeranyl tail of heme a3 has been removed for clarity. The blue arrow (H2O, H+ exit pathway) has been inserted to demonstrate the different relative orientation between Figures 1 and 2C. The figure was prepared with PyMOL.

Figure 1. Overall structure of T. thermophilus cytochrome ba3 oxidase (PDB accession code 3S8G). Subunits I, II and IIa are shown in the background as yellow, cyan and magenta cartoon, respectively. The four redox-active metal binding sites ((CuA−CuA), heme b, heme a3, CuB) are shown as sticks and the structurally resolved water molecules as red spheres. The four blue arrows represent the active, spatially routes followed by incoming substrates (O2 molecules, H+ ions), outcoming products (H2O molecules), and oxidizing metal cofactors (electrons), during the main catalytic function of the enzyme the reduction of O2 to H2O. The figure was prepared with PyMOL.

has been determined.4,11,12 It consists of three subunits with a total of 764 amino acid residues. The electron donor to cytochrome ba3 is cytochrome c552.4 The low-spin heme b and the binuclear heme a3-CuB active center presented in Figure 2, are contained in subunit I and the mixed valence homodinuclear (CuA1.5+-CuA1.5+) copper complex in subunit II. The a-type heme in cytochrome ba 3 contain a B


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Accounts of Chemical Research hydroxyethylgeranylgeranyl side chain instead of a hydroxyethylfarnesyl side chain as seen in most eukaryotic and bacterial oxidases.4,5 In addition, the highly conserved to all heme-copper oxidases Glu278 (residue numbering of Paracoccus denitrificans), that has been implicated in redox-induced proton transfer reactions is replaced by Ile in cytochrome ba3.4 The low-spin heme b is connected to the high-spin heme a3 of the binuclear center through His386, Phe385 and the axially coordinated to heme a3 Fe His384. The Fe-to-Fe distance between the two hemes is 13.9 Å and they approach each other at a short edge-to-edge distance of 5.0 Å and an angle of 110°. CuB is located 4.4 Å from heme a3 Fe and is coordinated by His282, His283 and His233, with the latter being covalently linked to Tyr237 residue.4 In this Account, we correlate the discrete ligand binding and electron transfer properties of cytochrome ba3 oxidase with an evolutionary adaption mechanism that must be present a priori in the extreme low oxygen and high temperature environments where cytochrome ba3 operates. We have applied Fourier transform Infrared (FTIR) and time-resolved step-scan FTIR spectroscopies (TRS2-FTIR) to probe the dynamics of the fully reduced (CuA1+ heme b2+ CuB1+ heme a32+) and the mixed valence (CuA2+ heme b3+ CuB1+ heme a32+) forms of the enzyme. Of significant importance is the property of selectively reducing the heme a3-CuB binuclear site by CO (2 e− reductant) that permits the measurement of the rate constants of individual electron transfer (ET) reactions such as the extent of the heme a3 Fe2+ to heme b Fe3+ reverse electron transfer induced by the photodissociation of CO and also the direct CuB1+ to mixed valence homodinuclear CuA1.5+−CuA1.5+ reverse ET. We report and analyze a series of protein factors that lead to the unique behavior of the enzyme. All the revealed and discussed factors are not independent, but highly interconnected, acting in a concerted way and leading to a thermostable, high O2-affinity enzyme.

Figure 3. Difference FTIR spectrum of fully reduced cytochrome ba3−CO complex at pD 8.50. Inset shows the same spectrum expanded by a factor of 10. Enzyme concentration was 1.5 mM, the optical path length 25 μm and the spectral resolution 4 cm−1. The total number of scans was 500.

The fact that at pD 8.5 some of the carboxylic residues are still protonated indicates that the combination of three factors described below influence the pKa value of the ionizable groups. (i) Dehydration/Born effect: The active site of cytochrome ba3 oxidase is buried into the hydrophobic core of the enzyme as evidenced by the lower H/D effect exhibited relative to other CcO. The presence of the structurally wellordered water molecules in the moiety of the propionates of heme a3 does not necessarily translate to a hydrophilic environment accessible to the solvent. (ii) Charge−charge Coulombic interactions: Cytochrome ba3 oxidase displays an unusual high pI of 10.4 (subunit I) with a + 9 net positive charge, favoring this way the deprotonation of Asp and Glu residues.16 (iii) Charge−dipole and dipole−dipole hydrogen bonding interactions: On the basis of the 3D structure,4,11 Asp372, Glu126 in subunit II and Asp287 could be the candidates for the protonated carboxylic residues revealed from the FTIR spectra. Asp372 is subjected to a tight Hbonding network with the carboxylate oxygen atoms in a 2.5 and 3.4 Å proximity to one of the oxygens of heme a3 pyrrole ring A propionate, to the confined Wat927 molecule of the socalled water pool at 2.9 Å, and also to His376 (3.8 Å) (Figure 2C). Asp287 and Glu126, belong to the same tight H-bond network around the water pool and above the heme a3 propionates, located at distances < 4.0 Å from Wat826, Wat940 and His376. Figure 2 also shows that heme b and heme a3 propionates are essentially protonated as evidenced from the mode detected at 1702 cm−1 and the abovementioned H-bond network. Therefore, the two hemes feel a net positive charge that favors electron transfer, since there is an increase in the driving force (ΔG). The net positive charge (+9) in subunit I is also documented by the unusual high pI of 10.4.16

2. PROTEIN ARCHITECTURE AND EVOLUTION Protein Structure in Cytochrome ba3 Oxidase

Figure 3 presents the equilibrium FTIR spectrum of the cytochrome ba3-CO complex in the 1500−1800 cm−1 spectral region at pD 8.50. In the inset, weak modes are detected in the 1700−1750 cm−1 region which are characteristic of the protonated propionates groups of the hemes and the carboxylic aspartates (Asp) or glutamates (Glu) residues.10 The 1702 cm−1 mode is assigned to the propionates of hemes b and a3, whereas the modes at 1717, 1734, and 1741 cm−1 to the protonated Asp and/or Glu residues.13 Cytochrome ba3 contains 26 Glu and 16 Asp residues out of a total of 764 residues.4 This corresponds to a 5.5% Glu and Asp content which is lower than the average 11.7% content observed in proteins.14 Similarly, cytochrome caa3 from T. thermophilus has also a low 6.3% of Glu and Asp content.15 We calculate that 12−14% protonated carboxylic residues are present at pD 8.5 and assuming an 80% CO character of the amide I band, its integrated intensity ratio relative to that of 1717, 1734, and 1741 cm−1 modes, suggests the presence of five to six protonated carboxylic residues in the cytochrome ba3−CO complex. In addition, the comparison of the intensity of the 1702 cm−1 mode with that of the carboxylic residues, indicates that all four propionates of hemes b and a3 are essentially protonated at pD 8.5.

Ligand Binding in the Fully Reduced (FR) and Mixed Valence (MV) Forms of Cytochrome ba3 Oxidase

Figure 4 shows the equilibrium FTIR spectrum of the fully reduced (FR) cytochrome ba3−CO complex at pD 8.5. Multiple heme a3 Fe2+−CO conformers are evident from the presence of CO sensitive modes at 1959.5, 1963.0, 1967.5, C


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Figure 5. Double difference FTIR spectrum of mixed valence [(CuA− CuA)3+, Feb3+, Fea32+, CuB1+] cytochrome ba3−12CO complex at pD 8.50. The inset shows the same spectrum expanded by a factor of 5. Same experimental conditions as in Figure 4.

Figure 4. Double difference FTIR spectrum of fully reduced [(CuA− CuA)2+, Feb2+, Fea32+, CuB1+] cytochrome ba3−12CO complex at pD 8.50. The inset shows the same spectrum expanded by a factor of 2.5. Same experimental conditions as in Figure 3. The total number of scans was 2000.

No additional CO sensitive modes, relative to the FR state, are detected and there is no difference after H/D exchange in the 6.5−8.5 pH/pD range. The ratio of the relative areas Fe−CO/ CuB−CO is reduced from 5.2 observed in the FR state to 4.7, but considering the change of the integrated absorptivities for the shifted heme a32+−CO and CuB1+−CO complexes (εFe−CO/εCu−CO = 1.62 from 1.68), we calculate a 71−29% equilibrium of the bound CO between the heme a3 Fe and CuB centers.13 The three major modes at 1971.0, 1975.5 and 1982.5 cm−1 represent the 92.8% of the total heme a3 Fe2+− CO complexes. The weak modes shown in the inset, observed at 1931.5 and 1938.0 cm−1 reflect the 1.1% natural abundance of 13CO and corresponds to the two major heme a3 Fe−13CO conformers. The 1982.5 cm−1 is the major conformer in expense of the 1975.5 cm−1 conformer. Figure 6 shows the FTIR spectra recorded during the formation of the MV cytochrome ba3−CO complex. In the first spectrum shown in black, it is evident that the ratio of the relative area Fea3−CO/CuB−CO is significantly reduced from that shown in Figure 5. At later times, the ratio Fea3−CO/ CuB−CO in the spectra shown in red, blue, and green is increased until the binuclear center is thermodynamically and kinetically equilibrated. The data suggest that the first station of CO in the binuclear center is the CuB atom and that formation of the heme a32+−CO complex follows subsequent to equilibration. A series of equilibration events also takes place between the three major heme a32+-CO conformers since in the first spectrum there is a statistical 50−50% initial distribution for the two main modes located at 1975.5 and 1982.5 cm−1. The equilibrium CuB1+−CO complex adopts a single conformation as evident in Figures 4 and 5, whereas the heme a3 Fe2+−CO complex appears to be in a fragile equilibrium of at least seven conformers. The CuB atom, in contrast to the heme a3, is subjected to a rigid and stable environment. The origin of the multiple Fe2+−CO modes, comes mainly from the existence of different conformations of the proximal side of heme a3, where its axial ligand His384 was found to have a significant larger Fe−N bond of 3.3 Å4, although later revisited to 2.2 Å in a high resolution structure of the protein.11 The variation in protonation state of the a3

1973.0, 1981.5, 1988.0, and 1994.0 cm−1, whereas the equilibrium CuB1+−CO moiety adopts a single conformation with a characteristic frequency at 2053.5 cm−1. The frequencies of the three major heme a3 Fe2+-CO bands are 10−20 cm−1 higher than those reported for other heme-copper oxidases,17−21 and close only to Acidianus ambivalens aa3 quinol oxidase observed at 1973 and 1977 cm−1.22 On the other hand, the CO-frequency of the equilibrium CuB1+-CO complex is 10−12 cm−1 lower than the corresponding modes of the transient CuB1+-CO complex reported for other heme-copper oxidases, and coincides with that observed at 2053 cm−1 in the B-type A. ambivalens aa3 quinol oxidase. All the observed modes showed no sensitivity to H/D exchange in the 5.25− 10.10 pH/pD range suggesting a highly rigid and hydrophobic environment for the binuclear center. The adoption of a single geometry for the CuB1+-CO complex reflects the stability of the CuB environment that involves His282, His283, and His233Tyr237, which is not subjected to protonation/deprotonation changes and/or structural fluctuations.13,23 The observed frequency shifts of the CO-bound modes and the inverted population ratio between the conformers at 1973.0 and 1981.5 cm−1, in the mixed valence form, are attributed to the presence of the catalytically formed CO2 molecules, from the reduction of the binuclear center by CO. The altered electrostatic interactions induced by the presence of the CO2 molecules in the binuclear center appear to shift the fragile 0.15 kcal/mol equilibrium. These observations are used to characterize the internal cavity where CO2 molecules reside and serve as a tracer of the local electrostatic interactions. In addition, the CO2 molecules are the products of a catalytic reaction, performed by the enzyme that has not been thoroughly studied yet. Therefore, it is important to follow the fate and the interactions exerted by the released CO2 molecules in the active center. Figure 5 presents the equilibrium FTIR spectrum of the mixed valence (MV) ba3−CO complex at pD 8.5. All the heme a3 Fe2+−CO modes detected in the FR state shown in Figure 4, are slightly upshifted by 1−3 cm−1 and the equilibrium CuB−CO mode is downshifted by 2.5 cm−1, in agreement with previous reports on E. coli bo319 and bovine aa3 oxidases.24,25 D


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formation of the CO complex in the MV state of the enzyme. For the first time, there is a direct spectroscopic evidence that CuB is the entry point of the active site and serves as a transient trap for the ligands present into the input channel.9,10,13,29 In the case of cytochrome ba3, this is of crucial importance, since the enzyme operates at high temperatures, where the solubility of O2 is reduced and the need of an efficient ligand-binding mechanism is further increased. Photodissociation of CO from the Fully Reduced (FR) and Mixed Valence (MV) Forms of Cytochrome ba3

Figure 7A presents the light-minus-dark difference FTIR spectrum of fully reduced (FR) cytochrome ba3−CO complex. Photolysis of the heme a3 Fe2+−CO complex by 416 nm continuous wave (CW) laser light results to the migration of CO to CuB1+.13,23,29 All seven heme a3−CO conformers present in the equilibrium FTIR spectrum displaced in Figure 4 are shown as negative peaks due to the photolysis of Fe-CO bond. The relative ratios of the Fe−CO bands are exactly the same as those observed in the equilibrium FTIR spectrum demonstrating that all conformers are photolabile. The equilibrium CuB−CO complex is not photolabile and remains as a spectator in the process.13 The frequency of the CO mode of the transient CuB−CO complex is the same with that observed for the equilibrium complex and no other CO modes are detected in the 2040−2070 cm−1 spectral region, even at a ratio of 0.5% of the 2053.5 cm−1 peak shown in Figure 7B.13,23 In Figure 8A, the light-minus-dark difference FTIR spectrum of the mixed valence (MV) form of ba3-CO complex is presented. The transient CuB1+-CO complex absorbs energy at the same frequency as the equilibrium complex at 2051.0 cm−1, slightly downshifted relative to the FR state which is observed at 2053.5 cm−1. The seven heme a3 Fe2+-CO modes appear as slightly upshifted negative peaks and their relative ratios are exactly the same with those observed in the equilibrium FTIR spectrum. As shown in Figure 8B, no other CO modes related to the CuB-CO complex are detected in the 2030−2070 cm−1 spectral region, even at a ratio of 0.5% of the 2051.0 cm−1

Figure 6. Double difference FTIR spectra monitoring the formation of the mixed valence cytochrome ba3−CO complex at pD 8.50. Spectra A, B, and C represent the nonequilibrated cytochrome ba3− CO complex taken at 2 h intervals subsequent to overnight incubation of the oxidized ba3 under CO, and spectrum D shows the final thermally equilibrated complex. Same experimental conditions as in Figure 4. The total number of scans was 500.

proximal heme Fe-His384 with Gly359 is responsible for the occurrence of the split Fe-His stretching mode, which has components at 193 and 210 cm−1.26 Furthermore, three resonance Raman (RR) studies collected evidence about relatively photostable five-coordinate (5C) heme a3 Fe2+−CO conformers, where the proximal ligand had been lost.26−28 Therefore, His384 appears to be weakly bound to Fe atom giving rise to a flexibility which may be important during the catalytic function of the enzyme. The binding of CO to cytochrome ba3 oxidase does not follow the general consensus found in other heme-copper oxidases. The high affinity of CuB for exogenous ligands demonstrated in Figures 4−6 allowed us to monitor the

Figure 7. (A) Light-minus-dark difference FTIR spectrum of the 12CO-bound form of fully reduced cytochrome ba3 oxidase at pD 8.50. (B) The same spectrum expanded by a factor of 7. Same experimental conditions as in Figure 4. The excitation wavelength was 416 nm and the incident power 13 mW. The photolysis yield was calculated to 35%. E


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Figure 8. (A) Light-minus-dark difference FTIR spectrum of the 12CO-bound form of mixed valence cytochrome ba3 oxidase at pD 8.50. (B) Same spectrum expanded by a factor of 6. Enzyme concentration was 1.5 mM, the path-length 25 μm, and the spectral resolution 4 cm−1. Same experimental conditions as in Figure 7.

photodissociation of CO from heme a3 of MV cytochrome ba3 as shown in Figure 8. This observation is in relative agreement with previous work based on optical spectroscopy measurements on cytochrome ba3−oxidase,10 but in contrast to all other aa3-, caa3- and bo3-oxidases examined, where back ET rates between 25 and 100% were observed.10,17−19,24 In contrast to its counterpart cytochrome caa3, cytochrome ba3 exhibits inverted potentials of 210 and 285/430 mV for the hemes b and a3, respectively.33 Both values are shifted to more positive potentials than other heme b containing proteins such as the sperm whale myoglobin (40 mV) and the heme a3 containing CcO (350 mV) of P. denitrificans aa3 oxidase. The observed shift to more positive potential is a result of increasing positive charge on the hemes. This can be achieved by protonation of the propionate groups of the heme(s), high hydrophobicity, and alterations of the heme(s) axial ligands. Indeed, subunit I of cytochrome ba3 is buried to an extreme hydrophobic region, having a pI of 10.4 and a net charge of + 9.16 The nonpolar, low-dielectric microenvironment does not allow the heme to receive electron density from water molecules. In addition, the present and previous reported spectroscopic data have shown that the propionates of hemes b and a3 are partially protonated in cytochrome ba3, in contrast to other A-type aa3 oxidases, and also that the heme a3-axial ligand His384 distance appears to be able to fluctuate, preventing a high degree of back-bonding electron donation to iron and favoring the electron entry to the active site.4,27,28 All of the above-mentioned parameters deviate from what it has been observed in other CcOs and have been tuned in such way to raise the positive charge sensed by the hemes. This leads to an increase of the midpoint potentials and stabilization of the reduced state of the hemes. During catalysis, heme a3 reduction is further favored due to the high driving force of the heme b-heme a3 ET step, as it was discussed above. Figure 9A shows the step-scan time-resolved FTIR (TRS2FTIR) difference spectra (td = 150 μs to 120 ms, 8 cm−1 spectral resolution) of the MV ba3−CO complex (pD 8.5) subsequent to CO-photolysis by a nanosecond laser pulse (532

peak. The absence of additional other than the CuB-CO mode at 2051.0 cm−1 in a spectral region with S/N ratio > 105 reflects the absence of back electron transfer from Fea32+ to [(CuA-CuA)3+, Feb3+] upon dissociation of CO from heme a3.13,23 If a [(CuA−CuA), Feb]5+, Fea33+, CuB1+−CO complex could be generated instead of the ordinary [(CuA−CuA), Feb]6+, Fea32+, CuB1+-CO mixed valence form, then the CuB−CO complex would exhibit a clear different CO stretching frequency. In particular, the CO mode would be shifted to lower frequencies since the more polar heme a3 Fe3+ would stabilize the Cδ+−Oδ− resonance form of the CO, leading to a reduced electron density into the π bonding molecular orbitals of CO. In bovine aa3 and E. coli bo3 oxidases which exhibit 25− 35% back electron transfer, the observed shifts of the transient CuB−CO complexes, caused by the reverse ET, were 2124 and 17 cm−1,19 respectively. In cytochrome ba3 the shift would be expected even larger since the heme a3 Fe-CuB distance is 4.4 Å which is shorter than that found in aa3, bo3, cbb3 and caa3 oxidases (4.8−5.3 Å). The data presented in Figures 7 and 8 reveal the high affinity of CuB for CO as evidenced in numerous previous studies.23,13,28−32 Due to the presence of CuB, the geminate rebinding of photolyzed CO to heme a3 is energetically inhibited due to the energy barrier of 10−15 kcal/mol, in contrast to 2.5 kcal/mol found in other typical heme proteins. Therefore, the photolyzed CO is transferred ballistically and 75−80% binds almost quantitatively to CuB.23 The slightly upshifted and downshifted heme a3 Fe2+−CO and CuB−CO modes, respectively, in the MV ba3−CO complex, reflect a lower electrostatic potential in the active site, relative to the FR state of the enzyme. The frequency of the νC‑O mode is sensitive to the electrostatic environment of the binding site leading to the observed opposite shifts since CO flips almost 180° in its way from heme a3 Fe2+ to CuB1+. The lack of additional, other than the CuB−CO mode at 2051.0 cm−1 in the 2030−2070 cm−1 spectral region suggests the absence of back electron transfer from heme a3 Fe2+ to heme b Fe3+, upon F


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an increased intensity at 1978.0 cm−1, signaling the rebinding of CO to heme a3. The ratio of the relative areas Fe−CO/ CuB−CO is 1.5 and remains constant for all data points, and thus, we conclude that no significant fraction of CO escapes the binuclear center at 293 K.23,30,31 The kinetic analysis displayed in Figure 9B suggests first order rates of 19.3 s−1 for the thermal release of CO from CuB and 17.3 s−1 for the rebinding to heme a3 Fe. These rates are 1.5 times smaller than those reported earlier for the FR ba3−CO complex.23,13 The data presented in Figure 9 support the general consensus revealed in previous step-scan studies with minor alterations.23,13 The photolyzed ligand is transferred almost ballistically to CuB and its thermal release is followed, as evidenced from the derived rates, by concomitant rebinding to heme a3. The decreased rates as compared to those earlier reported for the FR ba3−CO complex, are attributed to the slightly altered electrostatic potential of the MV binding site that leads, as evidenced in Figures 7 and 8, to a slightly higher affinity of CuB for CO. Therefore, the thermal release of CO becomes slower, the rate decreased by a factor of 1.2−1.5, and the rebinding to heme a3 is further energetically inhibited. Figure 9 further supports the absence of back ET between heme a3 Fe2+ and heme b Fe3+ upon photodissociation of CO, making the overall picture similar to that reported for the FR form. The CuB center is silent in the UV/vis region so its behavior cannot be monitored. In P. denitrificans CcO the calculated back ET was from 50% at pH 7 to 100% at pH 11. The subsequent CO recombination rate, was decreased by a factor of 2 at pH 7, and by a factor of 50 at pH 11 relative to the FR form.34 The decrease in the rate of CO recombination at alkaline conditions in the MV enzyme can be explained by a decrease of the occupancy of fully reduced binuclear site after photolysis due to electron transfer from heme a3 to heme a. Similar results were obtained for the bovine heart CcO where a 25−35% back ET was observed at pH 7.0−7.524 and in R. sphaeroides aa3CcO where a 100% back ET at pH 7.2 was reported.35 Since cytochrome ba3 does not exhibit back ET it is reasonable to expect minimal differences, relative to the FR form as those reported here. The CO rebinding is still determined by the thermal release of CO from CuB, which transiently binds almost quantitatively the photolyzed CO molecules.

Figure 9. (A) Time-resolved step-scan FTIR spectra of photodissociated mixed valence cytochrome ba3−CO complex (pD 8.50) at times between 0 and 120 ms subsequent to CO photolysis. The spectral resolution was 8 cm−1, and the time resolution was 100 μs. A 532 nm, 10 mJ/pulse pumpbeam was used for photolysis. (B) Kinetic analysis of the heme a32+-CO and CuB1+-CO modes. Shown is a plot of the 2051 cm−1 (squares) and 1977 cm−1 (circles) modes versus time subsequent to CO photolysis. ΔA was measured from the bands area at times between 0 and 120 ms subsequent to CO photolysis from heme a3. The curves are three-parameter exponential fits to the experimental data according to first-order kinetics.

3. EVOLUTIONARY ASPECTS: SUMMARY A detailed description of key issues related to electron transfer and proton pumping in the oxygen reductases of both the Afamily and the B-family have been reported.36 A plethora of data are available for the proton-pumping mechanisms for the A-family which require two proton input pathways (D- and Kchannels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the D-channel transporting all pumped protons. Cytochrome ba3 is member of the B-family, does not contain a D-channel and utilizes only one proton input channel, which is analogous to that of the Kchannel in the A-family, delivers protons to the active site for both O2 chemistry and proton pumping. In the B-family, mutations blocking the K-channel block both the oxidative and reductive reactions. The three K-channel mutants, Y248F, T312V, and E15A, although have little influence on the rate of reduction of heme b, each mutant prevents electron transfer from heme b to heme a3. In the A-family, mutations in the Kchannel have no influence on the four-electron oxidative

nm). Under these experimental conditions, the three major heme a3-CO conformers absorbing at 1971.0, 1975.5, and 1982.5 cm−1 could not be resolved, and thus, a single negative peak is observed at 1978.0 cm−1. The negative peak at 1978.0 cm−1 arises from the photolyzed heme a3−CO. The positive peak that appears at 2051.0 cm−1 is attributed to the CO stretch (νCO) of the transiently formed CuB1+−CO complex, as found under continuous light illumination,10 and arises from the near ballistic transfer of the photolyzed CO to CuB. Its intensity persists until 120 ms. The frequency of the CO mode in the transient CuB1+−CO complex is the same as that of the equilibrium CuB1+−CO. This observation suggests that no structural change at CuB occurs in association with CO binding to and dissociation from heme a3. No significant intensity variations are detected in the transient difference spectra (td = 5−3000 μs) for either the 2051.0 or 1978.0 cm−1 modes. At later times td = 3−120 ms, however, the decreased intensity of the transient 2051.0 cm−1 mode is accompanied by G


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of optimized forward ET which is a crucial point for the catalytic action. Cytochrome ba3 exhibits distinct and inverted redox potentials to ensure the arrival of electrons into the active site.33 This is mainly achieved by the high driving force ΔG° and secondarily through the low reorganization energy λ. The reversed order of the potentials, compared to A-type oxidases, guarantees the heme a-heme a3 intraprotein ET. Therefore, a complex network of cooperativities is not required in order to achieve exergonicity which is a mandatory requirement. Since electron and proton transfer are coupled, and cytochrome ba3 is not a fully effective proton pump, the need for optimized ET properties becomes essential. The finding that the three K-channel mutants Y248F, T312V, and E15A prevent electron transfer from heme b to heme a3 is consistent with the linear sequence of electron transfer occurring from CuA to heme b to heme a3 and demonstrates that the K-channel plays a role in the reductive half of the catalytic cycle. On this line based on spectroscopic, electrochemical, and theoretical studies on WT and single mutant CuA redox centers showed that thermal fluctuations may populate two alternative ground-state electronic wave functions optimized for electron entry and exit, respectively, through two different and nearly perpendicular pathways.37,38 These findings suggest a unique role for alternative electronic ground states indirectional electron transfer. The rate of reaction of the fully reduced enzyme with O2 demonstrated that both T312V and E15A mutations essentially prevent the oxidation reaction of both the heme a3 and heme b.

reaction but selectively inhibit the reduction of the active site before the reaction with the O2. The data presented here allow us to reach conclusions regarding the role of evolution in engineering cytochrome ba3 into a high oxygen affinity enzyme in order to adapt and function effectively in its extreme T = 47−85 °C environment. Based on the work from our laboratory in the field of CcOs and work from other laboratories, below, we outline important properties/functions of cytochrome ba3 oxidase and propose that when they are coupled, lead to a specific molecular mechanism able to ensure the effective thermodynamic and kinetic function of the enzyme. Rigidity and Nonpolar Hydrophobic Interactions

The active heme a3−CuB binuclear center is buried deep in the protein interior, in a region relatively inaccessible to solvent and governed by nonpolar hydrophobic interactions through the low water accessibility and small H/D frequency shifts of the observed CO marker bands. On the other hand, the wellordered, not exchangeable, structural water molecules detected in spectroscopic and crystallographic studies play significant role not only in the catalytic function of the enzyme, serving as possible proton acceptors, but also in the protein stability minimizing the desolvation energy penalty due to hydrophobicity. Especially, the water pool found in the proximity of the propionates of heme a3 in cytochrome ba3 can be viewed as a molecular variable able to define the internal wetting of the active center, and control thermodynamically and kinetically not only the chemistry, but also the stability of the enzyme. Different molecular mechanisms can be combined, leading in a variety of thermodynamic routes to thermal adaption. Usually, average hydrophobicity increases as optimal growth temperature increases preventing the solvent molecules from reaching protein interior. Internal hydration is also believed to be a major factor affecting the thermal stability of a protein. Changing hydration level in secondary binding sites (internal cavities) may lead to extensive structural fluctuations and different states that are able to play significant roles, such as thermodynamic and kinetic gating. Therefore, the hydrophobicity observed in cytochrome ba3, together with the tightly hydrogen-bonded detected structural waters around the water pool, appears to be part of the strategy employed by the thermophilic enzyme to defend against temperature.

Effective Proton-Input and Water/Proton-Exit Pathways (Proton-Loading Site)

Of profound importance is the identification of the water/ proton-exit pathways. In cytochrome ba3a protonic connectivity between the protonated propionates of heme a3 and protonated acidic residues Asp372, Asp287, and Glu126 which are located in the vicinity of the binuclear center has been demonstrated. These residues together with His283, Arg225, Arg449, Tyr133, Asn377, and His376 which are located close or next to heme a3, on the positive side of the membrane, form a hydrophilic cavity rich in waters (Figure 2C). Combining the present and previous data, the most reliable scenario involves a cluster of groups, water molecules and amino acid residues as the proton-loading site of cytochrome ba3 oxidase.36 Since this hydrophilic feature is structurally conserved among all the characterized CcOs, there is consensus that is the water/proton exit site.39−48

Ligand Input Channel and High Affinity of CuB for Exogenous Ligands

A hydrophobic ligand input channel is necessary for the operation of cytochrome ba3. At 70 °C and 1 bar atmospheric pressure, the solubility of O2 in water is 50% reduced and drops from ≈8.0 to ≈4.0 mg/L. Therefore, an input channel leading close to CuB ensures that the O2 molecules diffuse from the protein exterior to the active site. As previously stated a series of factors contribute to the overall behavior of the enzyme. Head-to-head comparison of the CuB site in various CcOs does not reveal a clear, significant structural difference that may be responsible for the high O2-affinity of CuB in cytochrome ba3. We attribute this behavior to the optimized hydrophobic O2-channel that leads directly above CuB, evolved or created by nature so as to ensure the unimpeded diffusion of the substrate to the active center.

Specifically Engineered Protein Structure and Flexibility

An electron withdrawing formyl group is present at C8 of heme a3 and a hydrophobic hydroxyethylgeranylgeranyl moiety at C2. Increased hydrophobicity might stabilize the heme a3 at high growth temperatures. Subunit I of cytochrome ba3 exhibits an unusual high pI of 10.4, providing evidence of an extreme hydrophobic environment with significant net positive charge. This correlates well with the findings presented in Figure 3 indicating protonated acidic groups and residues even at pH/pD 8.50. High pI values (net positive charge) do not lead directly to extreme hydrophobic environments. Due to charge−charge (Coulombic) interactions, the pKa values of all of the ionizable groups in a protein are decreased by a positively charged environment and increased by a negatively charged environment. In this work, we present evidence that acidic residues (Asp and Glu), in the vicinity of the binuclear center, remain protonated even at pD = 8.5. This can be

Optimized Intramolecular ET Pathways

The absence of reverse ET between CuB−CuA and heme a3− heme b indicates increased activation barriers and the existence H


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Accounts of Chemical Research Notes

explained through dehydration (Born effect) and charge− dipole (hydrogen bonding) interactions. When an ionizable group is buried in the interior of the protein (nonpolar, hydrophobic environment), where the dielectric constant is lower than that of water, the neutral form of the Asp and Glu residues is favored, so the pKa value is increased. In the case of cytochrome ba3, its active site is buried into the hydrophobic core of the enzyme as evidenced by the lower H/D effect exhibited relative to other oxidases. In addition, the pKa values of all of the ionizable groups in a protein are increased when hydrogen bonding is tighter to the protonated form, as found in the case of cytochrome ba3 where acidic residues (Asp372 and Asp287) and groups (heme a3 propionates) are involved into an extensive H-bond network (Figure 2C). The conclusion that “high pI provides evidence of an extreme hydrophobic environment” is specific for cytochrome ba3 and results through our data and the factors used to interpret them. Furthermore, the net positive charge has a favorable effect on the CuA-heme b and heme b-heme a3 ET processes, increasing the midpoint redox potentials and as a consequence the driving force ΔG. In the absence of the specific protein environment in cytochrome ba3, ET would still have been downhill as found in all the other CcOs. The total energy profile of the enzyme has been built through small structural alterations such as optimized O2 channel and ET that serve as compensations to the reduced proton pumping and low O2 environments. The ET processes of cytochrome ba 3 cannot be viewed independently, but rather as a part of the total adaption mechanism. The changes in redox potentials do not help with O2 binding at low partial pressures. They just optimize the electron flow to the binuclear center, avoiding the back ET leakage. Binding of O2 to heme is secured not only from direct thermodynamic point of view, but also from the high affinity of CuB, which in the case of cytochrome ba3 serves as the upgraded, more efficient, primary, and temporal stop for the exogenous ligands. All the above-described parameters are not independent, but highly interconnected, acting in a concerted way and leading to a high O2-affinity enzyme, through the observed discrete behavior of cytochrome ba3. The extreme hydrophobic environment of the cytochrome ba3 active center is believed to contribute to the increased thermostability, and the optimized O2-channel to the high O2-affinity. How these parameters are transferred and translated from ambient temperatures to the relevant extreme conditions, where cytochrome ba3 operates, remains an open question and is under investigation in our laboratory.

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The authors declare no competing financial interest. Biographies Constantinos Koutsoupakis received his Ph.D. in 2003 from the University of Crete, Greece. He is a special Teaching and Research staff in the Department of Environmental Science and Technology at Cyprus University of Technology. He is working in the field of Timeresolved FTIR spectroscopy of biological molecules. Tewfik Soulimane is currently Head of the Department of Chemical Sciences, University of Limerick. He is also Lead of the BioMaterials Cluster of the Bernal Institute. His research interests are membrane protein structure and function and biopiezoelectricity. Constantinos Varotsis received his Ph.D. in Chemical Physics from Michigan State University under the supervision of G. T. Babcock. He is currently Professor in the Department of Environmental Science and Technology at Cyprus University of Technology, Cyprus. He is working in the field of Biophysical Chemistry.

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ACKNOWLEDGMENTS We thank Dr. Eftychia Pinakoulaki at the University of Cyprus for stimulating discussion and for reading the manuscript. Financial support from Cyprus University of Technology is gratefully acknowledged.

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ABBREVIATIONS ATP, adenosine triphosphate; ET, electron transfer; FTIR, Fourier transform infrared; MCT, mercury cadmium telluride; FR, fully reduced; MV, mixed valence; CcO, cytochrome c oxidase; CW, continuous wave

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 00-357-2500-2451. Fax: 00-357-2500-2802. ORCID

Constantinos Koutsoupakis: 0000-0001-9301-1021 Constantinos Varotsis: 0000-0003-2771-8891 Funding

This work was supported by the Cyprus University of Technology research funds to C.V. and the Science Foundation Ireland BICF865 to T.S. I


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K


Discrete Ligand Binding and Electron Transfer Properties of

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