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Article

ns-μs Time-Resolved Step-Scan FTIR of ba3 Oxidoreductase from Thermus thermophilus: Protonic Connectivity of w941-w946-w927

1
Department of Environmental Science and Technology, Cyprus University of Technology, P.O. Box 50329, 3603 Lemesos, Cyprus
2
Chemical and Environmental Science Department and Materials & Surface Science Institute, University of Limerick, V94 T9PX Limerick, Ireland
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2016, 17(10), 1657; https://doi.org/10.3390/ijms17101657
Submission received: 15 August 2016 / Revised: 13 September 2016 / Accepted: 21 September 2016 / Published: 29 September 2016
(This article belongs to the Special Issue Computational Modelling of Enzymatic Reaction Mechanisms)

Abstract

:
Time-resolved step-scan FTIR spectroscopy has been employed to probe the dynamics of the ba3 oxidoreductase from Thermus thermophilus in the ns-μs time range and in the pH/pD 6–9 range. The data revealed a pH/pD sensitivity of the D372 residue and of the ring-A propionate of heme a3. Based on the observed transient changes a model in which the protonic connectivity of w941-w946-927 to the D372 and the ring-A propionate of heme a3 is described.

Graphical Abstract

1. Introduction

The electron and proton transfers in conjunction with the protonic connectivity between the environments sensed by key residues play a vital role in the biological function of proteins [1]. Τhe conformational rigidity of Thermophilic enzymes against heat denaturation has attracted the biotechnological research community because of the molecular events associated with enzymatic catalysis. Based on the crystal structure cytochrome ba3 from Thermus thermophilus contains a homodinuclear copper center (CuA), a low-spin heme b, and a heme a3-CuB center [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. Cytochrome ba3 catalyzes the reductions of oxygen (O2) to water (H2O) and of nitric oxide (NO) to nitrous oxide (N2O), as well and the oxidation of carbon monoxide (CO) to carbon dioxide (CO2) [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. The photolyzed ba3-CO species is an excellent model for time-resolved spectroscopic studies [7,8,9,13,15,18,24]. In the past, we used time-resolved Raman and step-scan FTIR (TRS2-FTIR) spectroscopy to probe the binding of CO to CuB and the structural changes of the ring-A propionate of heme a3 and D372 [7,8,9,10]. It was concluded that the trans/cis isomerization of the ring-D propionate plays a crucial role in controlling the orientations of “docked” CO between the heme rings-A and -D propionates and that the protein environment removes the barrier to the two orientations of CO [10]. The role of the heme a3-D372-H2O site and of ring-A propionate as proton carriers to the H2O pool, which is conserved among all structurally-known heme-copper oxidases, were reported [11,16,18,23]. The observation of deprotonated and protonated forms of heme a3 rings-A and -D propionate and D372 indicated a protonic connectivity between the ring-A propionate, a H2O molecule and D372. It was proposed that the environment of the ring-A heme a3 propionate-D372-H2O moiety can contribute to proton motion [18,23].
Time-resolved Raman and step-scan FTIR are powerful structure-sensitive techniques for exploring changes that occur to metal centers and individual amino acids as a result of changes in the ligation state of the metal centers and/or redox and conformational changes induced by the changes in the coordination of the metal centers [24,25,26,27]. The temperature dependency of these changes is expected to give insight into the thermostability of the thermophilic enzymes. Ligand photodissociation can also induce protonation/deprotonation reactions of key residues and the pH dependency of the photodynamic/protonation/deprotonation can contribute towards the elucidation of events not previously reached by other spectroscopic techniques. Furthermore, the detection of protonation/deprotonation of ionizable groups is important towards the elucidation of the proton motions that take place in cytochrome c oxidases. The dynamics of the protein cavities in controlling the motion of O2 migration to the binuclear heme Fe-CuB center is also important towards the elucidation of ligand binding since the enzyme operates at high temperature/low O2 concentration. To address these issues the Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) studies of the fully-reduced CO complex in the pH/pD 6–9 range were examined and compared to determine the conformations of the key residue D372 and those of the heme a3 ring-A propionate. The main goal was to compare the pH/pD results in a time-resolved approach for the protonated and deprotonated forms of ba3. The effect of H/D exchange and the dynamic behavior of the 1749/1743 and 1723 cm−1 modes which have been assigned to ν(COO(H)) of two conformations of the protonated forms of D372, as well as the coupling of the protonic connectivity of w941-w946-w927 to the ring-A propionate of heme a3 and D372 are discussed.

2. Results

Figure 1 shows the Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO at pH 7.0 subsequent to CO photolysis by a 7 ns 532 nm laser pulse. At td = 100 ns, the spectra show a peak at 1697 cm−1, “W” shape troughs at 1706 and 1724 cm−1, and also features at 1717(+), 1733(+), 1738 (−), 1744(−), and 1749(+) cm−1. The peak/trough at 1697/1706 cm−1 is characteristic of the perturbation of the C=O stretching band exhibiting stronger H-bonding interaction to surrounding groups in the transient spectra that we have assigned to the ring-A propionate of heme a3. [7]. At td = 500–80,000 ns the 1706 cm−1 mode appears as a doublet with intensity and frequency changes. The 1717 and 1733 cm−1 modes, which are not exhibiting frequency shifts or intensity changes in the td = 100–80,000 ns range, can be attributed to the C=O mode of either protonated aspartic or glutamic residues which are affected by the induced perturbation of CO photodissociation. The 1749/1738, 1744 cm−1 modes have been tentatively assigned to the ν(COO(H)) of two conformations of protonated D372 [7,18]. A broad negative mode at 1548 cm−1 is also shown and is tentatively assigned, in agreement with previous work, to originate from the coupled His-Tyr ring mode with large contributions from the C–N of the covalent bond between both ring systems [28]. The 1541 cm−1 positive mode appeared as a broad peak and it was attributed to amide II vibrations and remained unchanged in the td = 100–80,000 ns range [29]. Features consisting of a negative peak at 1530 and positive peak at 1559 cm−1 are present at td = 100 ns, and were previously assigned to the νas(COO) of the deprotonated form of the ring-A propionate of heme a3 [11,18,23]. This is evidence that there is equilibrium between the protonated and deprotonated forms of the ring-A propionate of heme a3. It should be noted that the transient binding of CO to CuB in aa3 oxidase is dynamically linked to structural changes around a protonated carboxyl group [30]. Finally, a peak/trough at 1506/1513 cm−1 is present and exhibits small intensity changes, but the ratio of the 1506/1513 cm−1 modes remained unchanged. This derivative form feature has been attributed to tyrosinate/tyrosine vibrations [31].
Figure 2 shows the Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO at pH 6.0 subsequent to CO photolysis by a 7 ns 532 nm laser pulse. The Time Resolved Step-Scan (TRS2) FTIR difference spectra in the 1690–1760 cm−1 region show the following changes when compared with those obtained at pH 7. At td = 100 ns, the protonated form of D372 is observed at 1742 cm−1, showing a 7 cm−1 downshift, which is representative of a weaker C=O bond exhibiting stronger H-bonding interaction to surrounding groups. The 1733 cm−1 mode has gained intensity, whereas that of the 1717 cm−1 remained the same. The 1697 cm−1 mode is not altered in intensity and/or frequency shifts; however, there are two weak negative peaks located at 1706 and 1714 cm−1, which at td = 80,000 ns have gained intensity and appeared as a single mode at 1714 cm−1. Compared to pH 7, we conclude that there is a pH sensitivity of the protonated forms of D372 and the ring-A propionate of heme a3. The observed 1728(−), 1733(+), and 1742(+) cm−1 modes do not present any intensity changes or frequency shifts in the td = 100–80,000 ns range. Compared to the pH 7 spectra, there is also a frequency shift of the 1723 cm−1 mode, which has been attributed to one of the two conformations of D372, to 1728 cm−1. This indicates sensitivity upon protonation of the second conformer of D372. The 1559 cm−1 mode is broader and the 1541 cm−1 mode at pH 6 is similar to that at pH 7. The negative peak at 1548 cm−1 observed at pH 7, becomes a doublet with the appearance of a new negative peak at 1554 cm−1. The trough at 1530 cm−1, which was previously assigned to the deprotonated form of νas(COO) of the ring-A propionate of heme a3, is present at pH 6 without presenting any changes regarded to the pH alteration.
Figure 3 shows the Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO at pH 9.0 subsequent to CO photolysis by a 7 ns 532 nm laser pulse. At td = 100 ns, the observed peak/trough feature of 1697/1706 cm−1 is similar to that observed at pH 7. This is in contrast to the pH 6 data where two negative peaks at 1706 and 1714 cm−1 were observed indicating the pH sensitivity of the ring-A propionate of heme a3. The protonated form of D372 observed at 1749 cm−1 exhibits a 7 cm−1 upshift when compared with that observed at pH 6, and a 3 cm−1 downshift when compared with that observed at pH 7, confirming the pH sensitivity of the protonated D372. In addition, the negative peak at 1739 cm−1, which has been assigned to D372, has gained intensity at td = 100 ns when compared to that at pH 9, but at td = 80 μs has lost almost all of its intensity. This observation indicates that the dynamics of the D372 are linked to the dynamics of the photodissociated CO [7,23]. The deprotonated forms of the ring-A propionate exhibit significant changes as the 1530 cm−1 mode appears as a doublet. In addition, at td = 100 ns, there are two trough at 1548 and 1554 cm−1. The latter trough loses intensity at times longer than 100 ns and disappears at td = 80 μs, indicating that its behavior is coupled to that of the 1739 cm−1 trough.
Figure 4 presents the Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of the fully-reduced ba3-CO complex in D2O. The experiments were performed in D2O in order to study the behavior of the protein upon H/D exchange. The amide I band arises 80% from the C=O stretching mode of the amide functional group and 20% from C–N stretching [8,9,10,11,12,13]. The protein secondary structure consists of a-helix (1648–1660 cm−1), β-sheet (1625–1640 and 1672–1694 cm−1), turns (1660–1685 cm−1), and unordered structures (1640–1650 cm−1) [32,33].
Figure 5 presents the pH sensitivity of Propionate A and aspartic acid residue D372. Features at 1736(+)/1744(−), 1729(−), 1697(+)/1706(−), 1686(+), 1668(+)/1675(−), 1652(+)/1660(−), 1638(+)/1644(−), 1630(−), 1559(+), 1541(+)/1548(−) 1519(−)/1527(−), 1535(−), and 1506(+)/1513(−) at 100 ns, subsequent to CO photolysis, remained unchanged in the td = 100–8000 ns range. The 1736(+)/1744(−) and 1729(−) features are slightly pH/pD-dependent since they show small frequency shifts, but the absence of the 1750 cm−1 in the pD spectra demonstrates the sensitivity of the protonated form of D372 to pH/pD exchanges. The 1697(+)/1706(−) feature, which has been attributed to the protonated form of the ring-A propionate, is insensitive to pD exchanges. A group of vibrations at 1668(+)/1675(−) are tentatively assigned to protein turns, those at 1652(+)/1660(−) to α-helical group of vibrations and, finally, those at 1638(+)/1644(−), 1630(−) to β-sheet [29,32]. All of the abovementioned vibrations remained unchanged in the td = 100–80,000 ns range. The behavior of all of the vibrational features observed at pD 7 are similar at at pD 6 and pD 9 (Figures S1 and S2).

3. Discussion

The Time Resolved Step-Scan FTIR data have already proven to be a very powerful for understanding the transient changes during protein action. The intensity/frequency changes observed in the TRS2-FTIR difference spectra is the result of the perturbation induced by the photodissociation of CO from heme a3 and its subsequent binding to CuB and to the docking site, which consists of the ring-A propionate heme a3-D372-H2O moiety. The presence of protonated/deprotonated forms of D372 and of the ring-A propionate, in association with the dependence of their deprotonated forms on the environment, indicates a protonic connectivity between the D372, the ring-A propionate of heme a3, and the pair of water molecules w941 and w927. To account for the presence of the observed pH/pD changes and the presence of protonated and deprotonated forms, we present, in Figure 6 and Figure 7, a scheme that includes the ring-A propionate/D372 pair and w927/941. In the oxidative or reductive phase, a proton can be accepted by the ring-A propionate/D372 pair, which influences the release of a proton to the H2O pool [34,35,36]. The w941 is not exchangeable; however, it contributes to the dynamics of the ring A-D372-w927. In the scheme, states B and D, in which a single proton is shared between the D372 and the heme a3 ring A-propionate, can accept a single proton. We propose that this is not operative in the protonated (A) or deprotonated (C) states. We postulate that the observed pH/pD changes in the TRS2-FTIR data are due to the exchangeable w927 that provides the H-bonded connection in the local moieties of the D372 and ring-A heme a3 propionate, and has activation energy for proton motion connecting the ligand docking site with the water pool. Consequently, during the formation of the chemical and pumped H+, the H2O pool may serve as a primary acceptor for the water molecules. The data reported here indicate that labile protons and w927 are the source of the observed changes to D372, whereas w946-w941-w942, with prop-A-D372 and His-376, form the proton loading site. The observation of H217O as a product in the reduction of the O2 reaction near H376, which is located in a complex with several crystallographically-detected H2O molecules, implies a unique H2O exit pathway [34]. At this point it should be noted that the mobility of H2O molecules in hydrophobic cavities makes them undetectable by X-ray crystallography. The ability of D2O to access the propionate-A-D372 moiety in the pD has been demonstrated by the observed changes to the frequencies of the protonated forms of propionate-A and D372 in the pD 6–9 range. It is suggested that w941/w946 in conjunction with Prop-A-H+ acts as the Zundel cation that forms the loading proton site (Figure 6 and Figure 7) [18]. In the absence of water molecules in the binuclear center we conclude that the proton loading site is located in the heme a3 Prop-A-w946-w941w927-D372 moiety [37].

4. Materials and Methods

4.1. Sample Preparation

Cytochrome ba3 was isolated from Thermus thermophilus HB8 cells according to previously published procedures. The ba3 samples were placed in a desired 0.1 M buffer, pH/pD 7.0, HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid), pH/pD 6.0, MES hydrate (2-(N-morpholino) ethanesulfonic acid hydrate, 4-morpholineethanesulfonic acid) and pH/pD 9.0, CHES (2-(cyclohexylamino)ethanesulfonic acid). The buffers prepared for the D2O experiments were measured assuming pD = pH(observed) + 0.4. The concentration of the samples was determined by UV-VIS measurements performed on a Lambda 25 UV-VIS spectrometer (Perkin Elmer, Italy), using ε416,ox = 152 mM−1·cm−1, and was ~700 μM. The fully-reduced CO bound form of the enzyme (ba3-CO) was prepared by using sodium dithionite as a reducing agent and subsequently exposed to 1 atm of CO under anaerobic conditions. The final samples were transferred to an air-tight, sealed FTIR cell, composed by two CaF2 windows. The path length was 6 μm for the samples in H216O and 15 μm for the samples in D2O. The total enzyme volume used for the experiments was ~1.5 mL. The 12CO gas was obtained from Messer (Germany) and D2O was purchased from Sigma-Aldrich (Taufkirchen, Germany).

4.2. ns-μs Time-Resolved Step-Scan FTIR Spectroscopy

The ns-μs Time Resolved Step-Scan Fourier Transform Infrared measurements (TRS2-FTIR) were performed on a Vertex 70 v FTIR spectrometer (Bruker, Karlsruhe, Germany) fitted with a liquid nitrogen-cooled fast Mercury-Cadmium-Telluride (MCT) detector (Figure 8). The optical bench was kept under vacuum conditions and the sample compartment was purged with N2. The spectral resolution was 4 cm−1 and the time resolution was 100 ns. The covered spectral range was 1200–2400 cm−1 and an Infrared filter 4200 nm (Spectrogon US INC., Mountain Lakes, NJ, USA) was used. The total number of time slices was 800; 50 of them were taken before the laser triggering and were used as a background reference for the data analysis, and 750 time slices were taken after laser triggering. A 532 nm laser pulse (second harmonic) from a Continuum Minilite Nd-YAG laser (Continuum, San Jose, CA, USA) (7 ns width, 5–8 mJ/pulse, 8 Hz) was used to photolyze the heme a3-CO complex. Two mirrors were used to direct the 532 nm laser beam inside the spectrometer and through the sample. A Quantum Composers Plus pulse delay generator, Model 9514 (Quantum Composers Inc., Bozeman Montana, MT, USA) was used to synchronize the spectrometer with the laser. A total of 10 coadditions per retardation data point were collected and 35 measurements of single-sided interferograms were collected and averaged in order to improve the S/N ratio. The AC and DC measurements were taken separately using the same sample. The AC signal was amplified by a factor of two using a Model SR560 Low-Noise preamplifier (Stanford research systems, Sunnyvale, CA, USA). The phase from DC measurements was used for phase correction of the AC measurements. The Blackman–Harris three-term apodization function with 32-cm−1 phase resolution and the Mertz/No Peak search phase correction algorithm were used. Difference spectra were calculated using ΔA = −log (IS/IR).

Supplementary Materials

Supplementary materials can be found at www.mdpi.com/1422-0067/17/10/1657/s1.

Acknowledgments

This work was supported by funds from the Cyprus Research. Promotion Foundation to C.V. (TEXNOLOGIA/THEPIS/0609(BE)/05.

Author Contributions

Antonis Nicolaides performed the experiments and analyzed the data; Tewfik Soulimane provided materials and Constantinos Varotsis wrote the paper.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 7.0.
Figure 1. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 7.0.
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Figure 2. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 6.0.
Figure 2. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 6.0.
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Figure 3. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 9.0.
Figure 3. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 9.0.
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Figure 4. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pD 7.0.
Figure 4. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pD 7.0.
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Figure 5. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1690–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 6.0, 7.0, and 9.0.
Figure 5. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of 1690–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 6.0, 7.0, and 9.0.
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Figure 6. The binuclear heme a3-CuB center and region of the heme a3 propionates of ba3 oxidoreductase from Thermus thermophilus illustrating the residues of interest [6]. Red, yellow and blue colors represent the oxygen, carbon and nitrogen atoms, respectively. The blue sphere represents the CuB atom. In w941, w946 and w927 the red and blue colors represent the oxygen and hydrogen atoms, respectively. The highlighted water molecules are conserved in heme-copper oxidases [18].
Figure 6. The binuclear heme a3-CuB center and region of the heme a3 propionates of ba3 oxidoreductase from Thermus thermophilus illustrating the residues of interest [6]. Red, yellow and blue colors represent the oxygen, carbon and nitrogen atoms, respectively. The blue sphere represents the CuB atom. In w941, w946 and w927 the red and blue colors represent the oxygen and hydrogen atoms, respectively. The highlighted water molecules are conserved in heme-copper oxidases [18].
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Figure 7. Protonic connectivity between the ring-A propionate of heme a3, the D372, and the water molecule w927. Blue, red and yellow colors represent protons, oxygen and carbon atoms, respectively. In states B and D, a single proton is shared between ring-A propionate of heme a3 and D372, while in state A, ring-A propionate of heme a3 and D372 are protonated and in state C, ring-A propionate of heme a3 and D372 are deprotonated.
Figure 7. Protonic connectivity between the ring-A propionate of heme a3, the D372, and the water molecule w927. Blue, red and yellow colors represent protons, oxygen and carbon atoms, respectively. In states B and D, a single proton is shared between ring-A propionate of heme a3 and D372, while in state A, ring-A propionate of heme a3 and D372 are protonated and in state C, ring-A propionate of heme a3 and D372 are deprotonated.
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Figure 8. Experimental setup for the ns Time-Resolved Step-Scan Fourier Transform Infrared (ns TRS2−FTIR). The red and green arrows represent the infrared beam and the 532 nm photolysis beam, respectively.
Figure 8. Experimental setup for the ns Time-Resolved Step-Scan Fourier Transform Infrared (ns TRS2−FTIR). The red and green arrows represent the infrared beam and the 532 nm photolysis beam, respectively.
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Nicolaides, A.; Soulimane, T.; Varotsis, C. ns-μs Time-Resolved Step-Scan FTIR of ba3 Oxidoreductase from Thermus thermophilus: Protonic Connectivity of w941-w946-w927. Int. J. Mol. Sci. 2016, 17, 1657. https://doi.org/10.3390/ijms17101657

AMA Style

Nicolaides A, Soulimane T, Varotsis C. ns-μs Time-Resolved Step-Scan FTIR of ba3 Oxidoreductase from Thermus thermophilus: Protonic Connectivity of w941-w946-w927. International Journal of Molecular Sciences. 2016; 17(10):1657. https://doi.org/10.3390/ijms17101657

Chicago/Turabian Style

Nicolaides, Antonis, Tewfik Soulimane, and Constantinos Varotsis. 2016. "ns-μs Time-Resolved Step-Scan FTIR of ba3 Oxidoreductase from Thermus thermophilus: Protonic Connectivity of w941-w946-w927" International Journal of Molecular Sciences 17, no. 10: 1657. https://doi.org/10.3390/ijms17101657

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