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

Nicolaides, Antonis; Soulimane, Tewfik; Varotsis, Constantinos

doi:10.3390/ijms17101657

Cited by 6 publications

(3 citation statements)

References 37 publications

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“…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 ba 3 oxidase . Since this hydrophilic feature is structurally conserved among all the characterized C c Os, there is consensus that is the water/proton exit site. − …”

Section: Evolutionary Aspects: Summarysupporting

confidence: 56%

Discrete Ligand Binding and Electron Transfer Properties of ba₃-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments

2019

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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 ba 3 oxidase is a B-type 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 ba 3 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 ba 3 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 ba 3 oxidase into a high O2-affinity enzyme that functions effectively in its extreme native environment. The present results show that ba 3-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 ba 3 oxidase.

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Section: Evolutionary Aspects: Summarysupporting

confidence: 56%

Discrete Ligand Binding and Electron Transfer Properties of ba₃-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments

2019

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“…Because of the evident benefits of light as a perturbation agent, important efforts have been invested to develop strategies to “activate” naturally light-insensitive proteins with light. For instance, light has been used to induce the release of heme-bound CO, monitoring its subsequent rebinding in myoglobin, , hemoglobin, O 2 -sensing proteins, , or cytochrome c oxidase, − or its replacement by O 2 to start the catalytic cycle of cytochrome c oxidase. ,, Light can also be used to study light-insensitive proteins with redox centers by means of external photoexcitable electron donors, such as flavin mononucleotide or ruthenium complexes. , Finally, the release of molecules can be induced with light using caged compounds, − allowing study of the dynamics and mechanism of ligand binding to receptors or of enzymatic reactions. ,, …”

Section: Perturbation Methodsmentioning

confidence: 99%

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

2020

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Infrared difference spectroscopy probes vibrational changes of proteins upon their perturbation. Compared with other spectroscopic methods, it stands out by its sensitivity to the protonation state, H-bonding, and the conformation of different groups in proteins, including the peptide backbone, amino acid side chains, internal water molecules, or cofactors. In particular, the detection of protonation and H-bonding changes in a timeresolved manner, not easily obtained by other techniques, is one of the most successful applications of IR difference spectroscopy. The present review deals with the use of perturbations designed to specifically change the protein between two (or more) functionally relevant states, a strategy often referred to as reaction-induced IR difference spectroscopy. In the first half of this contribution, I review the technique of reaction-induced IR difference spectroscopy of proteins, with special emphasis given to the preparation of suitable samples and their characterization, strategies for the perturbation of proteins, and methodologies for time-resolved measurements (from nanoseconds to minutes). The second half of this contribution focuses on the spectral interpretation. It starts by reviewing how changes in H-bonding, medium polarity, and vibrational coupling affect vibrational frequencies, intensities, and bandwidths. It is followed by band assignments, a crucial aspect mostly performed with the help of isotopic labeling and site-directed mutagenesis, and complemented by integration and interpretation of the results in the context of the studied protein, an aspect increasingly supported by spectral calculations. Selected examples from the literature, predominately but not exclusively from retinal proteins, are used to illustrate the topics covered in this review.

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“…Although the identity of PLS was proposed [ 17 , 18 , 19 , 20 ], there was no attempt to directly observe its protonation/deprotonation status. One of the methods that allows protolytic transitions to be shown is Fourier transform infrared (FTIR) spectroscopy, and particularly time-resolved step-scan FTIR (TRS 2 -FTIR) spectroscopy [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. FTIR spectroscopy was successfully applied to cytochrome c oxidase (e.g., [ 27 , 28 ]) and many other enzymes such as Ca 2+ ATPase [ 32 ]), light-induced enzymes [ 33 , 34 ], bacteriorhodopsin [ 35 ], and redox induced enzymes such as respiratory Complex I [ 36 ], cytochrome bd [ 37 ], and bc 1 [ 38 ], including time-resolved FTIR studies, for example, on channelrhodopsin-2 [ 39 ], bacteriorhodopsin [ 40 , 41 ], photosystem II [ 42 ], and cytochrome oxidase ba 3 [ 30 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy

2020

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Crystal structure analyses at atomic resolution and FTIR spectroscopic studies of cytochrome c oxidase have yet not revealed protonation or deprotonation of key sites of proton transfer in a time-resolved mode. Here, a sensitive technique to detect protolytic transitions is employed. In this work, probing a proton-loading site of cytochrome c oxidase from Paracoccus denitrificans with time-resolved Fourier transform infrared spectroscopy is presented for the first time. For this purpose, variants with single-site mutations of N131V, D124N, and E278Q, the key residues in the D-channel, were studied. The reaction of mutated CcO enzymes with oxygen was monitored and analyzed. Seven infrared bands in the “fast” kinetic spectra were found based on the following three requirements: (1) they are present in the “fast” phases of N131V and D124N mutants, (2) they have reciprocal counterparts in the “slow” kinetic spectra in these mutants, and (3) they are absent in “fast” kinetic spectra of the E278Q mutant. Moreover, the double-difference spectra between the first two mutants and E278Q revealed more IR bands that may belong to the proton-loading site protolytic transitions. From these results, it is assumed that several polar residues and/or water molecule cluster(s) share a proton as a proton-loading site. This site can be propionate itself (holding only a fraction of H+), His403, and/or water cluster(s).

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

Cited by 6 publications

References 37 publications

Discrete Ligand Binding and Electron Transfer Properties of ba₃-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments

Discrete Ligand Binding and Electron Transfer Properties of ba₃-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy

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

Cited by 6 publications

References 37 publications

Discrete Ligand Binding and Electron Transfer Properties of ba3-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments

Discrete Ligand Binding and Electron Transfer Properties of ba3-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy

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Discrete Ligand Binding and Electron Transfer Properties of ba₃-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments

Discrete Ligand Binding and Electron Transfer Properties of ba₃-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments