BBA - Bioenergetics (v.1707, #1)

Preface (vi-vii).

More than 50 years ago, initial experiments on enzymatic photorepair of ultraviolet (UV)-damaged DNA were reported [Proc. Natl. Acad. Sci. U. S. A. 35 (1949) 73]. Soon after this discovery, it was recognized that one enzyme, photolyase, is able to repair UV-induced DNA lesions by effectively reversing their formation using blue light. The enzymatic process named DNA photoreactivation depends on a non-covalently bound cofactor, flavin adenine dinucleotide (FAD). Flavins are ubiquitous redox-active catalysts in one- and two-electron transfer reactions of numerous biological processes. However, in the case of photolyase, not only the ground-state redox properties of the FAD cofactor are exploited but also, and perhaps more importantly, its excited-state properties. In the catalytically active, fully reduced redox form, the FAD absorbs in the blue and near-UV ranges of visible light. Although there is no direct experimental evidence, it appears generally accepted that starting from the excited singlet state, the chromophore initiates a reductive cleavage of the two major DNA photodamages, cyclobutane pyrimidine dimers and (6–4) photoproducts, by short-distance electron transfer to the DNA lesion. Back electron transfer from the repaired DNA segment is believed to eventually restore the initial redox states of the cofactor and the DNA nucleobases, resulting in an overall reaction with net-zero exchanged electrons. Thus, the entire process represents a true catalytic cycle.Many biochemical and biophysical studies have been carried out to unravel the fundamentals of this unique mode of action. The work has culminated in the elucidation of the three-dimensional structure of the enzyme in 1995 that revealed remarkable details, such as the FAD-cofactor arrangement in an unusual U-shaped configuration. With the crystal structure of the enzyme at hand, research on photolyases did not come to an end but, for good reason, intensified: the geometrical structure of the enzyme alone is not sufficient to fully understand the enzyme's action on UV-damaged DNA. Much effort has therefore been invested to learn more about, for example, the geometry of the enzyme–substrate complex, and the mechanism and pathways of intra-enzyme and enzyme ↔DNA electron transfer. Many of the key results from biochemical and molecular biology characterizations of the enzyme or the enzyme–substrate complex have been summarized in a number of reviews. Complementary to these articles, this review focuses on recent biophysical studies of photoreactivation comprising work performed from the early 1990s until the present.
Keywords: DNA repair; Flavin adenine dinucleotide; Cyclobutane pyrimidine dimer; (6–4) Photoproduct; Electron transfer; Radical-pair mechanism; Superexchange;

Quantum chemical methods are today a viable tool in the study of enzyme catalysis. The development of new density functional techniques and the enormous advancement in computer power have made it possible to accurately describe active sites of enzymes. This review gives a brief account of the methods and models used in this field. Three specific enzymes are discussed: pyruvate-formate lyase (PFL), spore photoproduct lyase (SPL), and benzylsuccinate synthase (BSS). What these enzymes have in common is that they use radical chemistry to catalyze C–C bond formation or cleavage reactions.
Keywords: Density functional theory; Pyruvate-formate lyase; Spore photoproduct lyase; Benzylsuccinate synthase; Radical enzyme;

Several novel enzyme reactions have recently been discovered in the aromatic metabolism of anaerobic bacteria. Many of these reactions appear to be catalyzed by oxygen-sensitive enzymes by means of highly reactive radical intermediates. This contribution deals with two key reactions in this metabolism: the ATP-driven reductive dearomatisation of the benzene ring and the reductive removal of a phenolic hydroxyl group. The two reactions catalyzed by benzoyl-CoA reductase (BCR) and 4-hydroxybenzoyl-CoA reductase (4-HBCR) are both mechanistically difficult to achieve; both are considered to proceed in ‘Birch-like’ reductions involving single electron and proton transfer steps to the aromatic ring. The problem of both reactions is the extremely high redox barrier for the first electron transfer to the substrate (e.g., −1.9 V in case of a benzoyl-CoA (BCoA) analogue), which is solved in the two enzymes in different manners. Studying these enzymatic reactions provides insights into general principles of how oxygen-dependent reactions are replaced by alternative processes under anoxic conditions.
Keywords: Enzyme; Anaerobic; Birch-like;

Tyrosine radicals play catalytic roles in essential metalloenzymes. Their properties—midpoint potential, stability…—or environment varies considerably from one enzyme to the other. To understand the origin of these properties, the redox tyrosines are studied by a number of spectroscopic techniques, including Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopy. An increasing number of vibrational data are reported for the (modified-) redox active tyrosines in ribonucleotide reductases, photosystem II, heme catalase and peroxidases, galactose and glyoxal oxidases, and cytochrome oxidase. The spectral markers for the tyrosinyl radicals have been recorded on models of (substituted) phenoxyl radicals, free or coordinated to metals. We review these vibrational data and present the correlations existing between the vibrational modes of the radicals and their properties and interactions formed with their environment: we present that the ν7a(C–O) mode of the radical, observed both by RR and FTIR spectroscopy at 1480–1515 cm−1, is a sensitive marker of the hydrogen bonding status of (substituted)-phenoxyl and Tyr, while the ν8a(C–C) mode may probe coordination of the Tyr to a metal. For photosystem II, the information obtained by light-induced FTIR difference spectroscopy for the two redox tyrosines TyrD and TyrZ and their hydrogen bonding partners is discussed in comparison with those obtained by other spectroscopic methods.
Keywords: Photosystem II; Tyrosinyl radical; Phenoxyl radical; Fourier transform infrared (FTIR) spectroscopy; Resonance Raman; Metallo-radical enzymes;

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron–electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.
Keywords: Ribonucleotide reductase; High-field EPR; ENDOR; Tyrosyl; Tryptophan; Cysteine radical;

EPR studies on radical enzymes are reviewed under the aspects of the information that they can provide and of the techniques that are used. An overview of organic radicals derived from amino acids, modified amino acids, and cofactors is given and g tensor data are compiled. The information accessible from a spectroscopic point of view is contrasted with the information required to understand enzyme structure and function, and some precautions are discussed that must be taken to derive the latter kind of information from the former. Structural dynamics is identified as an aspect that has rarely been addressed in the past although it is highly relevant for enzyme function. It is proposed that techniques introduced recently on other classes of proteins could help to close this gap.
Keywords: EPR; ENDOR; Tyrosyl radical; Glycyl radical; Ribonucleotide reductase; Cob(II)alamine;

Exploring amino-acid radical chemistry: protein engineering and de novo design by Kristina Westerlund; Bruce W. Berry; Heidi K. Privett; Cecilia Tommos (103-116).
Amino-acid radical enzymes are often highly complex structures containing multiple protein subunits and cofactors. These properties have in many cases hampered the detailed characterization of their amino-acid redox cofactors. To address this problem, a range of approaches has recently been developed in which a common strategy is to reduce the complexity of the radical-containing system. This work will be reviewed and it includes the light-induced generation of aromatic radicals in small-molecule and peptide systems. Natural redox proteins, including the blue copper protein azurin and a bacterial photosynthetic reaction center, have been engineered to introduce amino-acid radical chemistry. The redesign strategies to achieve this remarkable change in the properties of these proteins will be described. An additional approach to gain insights into the properties of amino-acid radicals is to synthesize de novo designed model proteins in which the redox chemistry of these species can be studied. Here we describe the design, synthesis and characteristics of monomeric three-helix bundle and four-helix bundle proteins designed to study the redox chemistry of tryptophan and tyrosine. This work demonstrates that de novo protein design combined with structural, electrochemical and quantum chemical analyses can provide detailed information on how the protein matrix tunes the thermodynamic properties of tryptophan.
Keywords: De novo protein design; Maquette; Protein radical; Tyrosyl radical; Tryptophanyl radical; Redox-active amino acid;

Transient radical pairs studied by time-resolved EPR by Robert Bittl; Stefan Weber (117-126).
Photogenerated short-lived radical pairs (RP) are common in biological photoprocesses such as photosynthesis and enzymatic DNA repair. They can be favorably probed by time-resolved electron paramagnetic resonance (EPR) methods with adequate time resolution. Two EPR techniques have proven to be particularly useful to extract information on the working states of photoinduced biological processes that is only difficult or sometimes even impossible to obtain by other types of spectroscopy. Firstly, transient EPR yields crucial information on the chemical nature and the geometry of the individual RP halves in a doublet-spin pair generated by a short laser pulse. This time-resolved method is applicable in all magnetic field/microwave frequency regimes that are used for continuous-wave EPR, and is nowadays routinely utilized with a time resolution reaching about 10 ns. Secondly, a pulsed EPR method named out-of-phase electron spin echo envelope modulation (OOP-ESEEM) is increasingly becoming popular. By this pulsed technique, the mutual spin–spin interaction between the RP halves in a doublet-spin pair manifests itself as an echo modulation detected as a function of the microwave-pulse spacing of a two-pulse echo sequence subsequent to a laser pulse. From the dipolar coupling, the distance between the radicals is readily derived. Since the spin–spin interaction parameters are typically not observable by transient EPR, the two techniques complement each other favorably. Both EPR methods have recently been applied to a variety of light-induced RPs in photobiology. This review summarizes the results obtained from such studies in the fields of plant and bacterial photosynthesis and DNA repair mediated by the enzyme DNA photolyase.
Keywords: Transient EPR; Ouf-of-phase electron spin echo envelope modulation; Radical pair; Photosynthesis; Photolyase;

The reaction between hydroperoxides and the haem group of proteins and enzymes is important for the function of many enzymes but has also been implicated in a number of pathological conditions where oxygen binding proteins interact with hydrogen peroxide or other peroxides. The haem group in the oxidized Fe3+ (ferric) state reacts with hydroperoxides with a formation of the Fe4+=O (oxoferryl) haem state and a free radical primarily located on the π-system of the haem. The radical is then transferred to an amino acid residue of the protein and undergoes further transfer and transformation processes. The free radicals formed in this reaction are reviewed for a number of proteins and enzymes. Their previously published EPR spectra are analysed in a comparative way. The radicals directly detected in most systems are tyrosyl radicals and the peroxyl radicals formed on tryptophan and possibly cysteine. The locations of the radicals in the proteins have been reported as follows: Tyr133 in soybean leghaemoglobin; αTyr42, αTrp14, βTrp15, βCys93, (αTyr24−αHis20), all in the α- and β-subunits of human haemoglobin; Tyr103, Tyr151 and Trp14 in sperm whale myoglobin; Tyr103, Tyr146 and Trp14 in horse myoglobin; Trp14, Tyr103 and Cys110 in human Mb. The sequence of events leading to radical formation, transformation and transfer, both intra- and intermolecularly, is considered. The free radicals induced by peroxides in the enzymes are reviewed. Those include: lignin peroxidase, cytochrome c peroxidase, cytochrome c oxidase, turnip isoperoxidase 7, bovine catalase, two isoforms of prostaglandin H synthase, Mycobacterium tuberculosis and Synechocystis PCC6803 catalase-peroxidases.
Keywords: Haem; Heme; Peroxide; Tyrosine; Tryptophan; Cysteine; Radical;