Cecilia Tommos

Proton and hydrogen currents in photosynthetic water oxidation

The photosynthetic processes that lead to water oxidation involve an evolution in time from photon dynamics to photochemically-driven electron transfer to coupled electron/proton chemistry. The redox-active tyrosine, Y(Z), is the component at which the proton currents necessary for water oxidation are switched on. The thermodynamic and kinetic implications of this function for Y(Z) are discussed. These considerations also provide insight into the related roles of Y(Z) in preserving the high photochemical quantum efficiency in Photosystem II (PSII) and of conserving the highly oxidizing conditions generated by the photochemistry in the PSII reaction center. The oxidation of Y(Z) by P(680)(+) can be described well by a treatment that invokes proton coupling within the context of non-adiabatic electron transfer. The reduction of Y(.)(Z), however, appears to proceed by an adiabatic process that may have hydrogen-atom transfer character.

A hydrogen-atom abstraction model for the function of YZ in photosynthetic oxygen evolution

Recent magnetic-resonance work on YŻ suggests that this species exhibits considerable motional flexibility in its functional site and that its phenol oxygen is not involved in a well-ordered hydrogen-bond interaction (Tang et al., submitted; Tommos et al., in press). Both of these observations are inconsistent with a simple electron-transfer function for this radical in photosynthetic water oxidation. By considering the roles of catalytically active amino acid radicals in other enzymes and recent data on the water-oxidation process in Photosystem II, we rationalize these observations by suggesting that YŻ functions to abstract hydrogen atoms from aquo- and hydroxy-bound managanese ions in the (Mn)4 cluster on each S-state transition. The hydrogen-atom abstraction process may occur either by sequential or concerted kinetic pathways. Within this model, the (Mn)4/YZ center forms a single catalytic center that comprises the Oxygen Evolving Complex in Photosystem II.

Manganese and tyrosyl radical function in photosynthetic oxygen evolution

Photosystem II catalyzes the photosynthetic oxidation of water to O2. The structural and functional basis for this remarkable process is emerging. The catalytic site contains a tetramanganese cluster, calcium, chloride and a redox-active tyrosine organized so as to promote electroneutral hydrogen atom abstraction from manganese-bound substrate water by the tyrosyl radical. Recent work is assessed within the framework of this model for the water oxidizing process.

Reversible voltammograms and a Pourbaix diagram for a protein tyrosine radical

Reversible voltammograms and a voltammetry half-wave potential versus solution pH diagram are described for a protein tyrosine radical. This work required a de novo designed tyrosine-radical protein displaying a unique combination of structural and electrochemical properties. The α3Y protein is structurally stable across a broad pH range. The redox-active tyrosine Y32 resides in a desolvated and well-structured environment. Y32 gives rise to reversible square-wave and differential pulse voltammograms at alkaline pH. The formal potential of the Y32-O•/Y32-OH redox couple is determined to 918 ± 2 mV versus the normal hydrogen electrode at pH 8.40 ± 0.01. The observation that Y32 gives rise to fully reversible voltammograms translates into an estimated lifetime of ≥30 ms for the Y32-O• state. This illustrates the range of tyrosine-radical stabilization that a structured protein can offer. Y32 gives rise to quasireversible square-wave and differential pulse voltammograms at acidic pH. These voltammograms represent the Y32 species at the upper edge of the quasirevesible range. The square-wave net potential closely approximates the formal potential of the Y32-O•/Y32-OH redox couple to 1,070 ± 1 mV versus the normal hydrogen electrode at pH 5.52 ± 0.01. The differential pulse voltammetry half-wave potential of the Y32-O•/Y32-OH redox pair is measured between pH 4.7 and 9.0. These results are described and analyzed.

Concerted hydrogen-atom abstraction in photosynthetic water oxidation

Photosystem II evolves oxygen by using water in the unlikely role of a reductant. The absorption of sunlight by chlorophyll produces highly oxidizing equivalents that are filled with electrons stripped from water. This proton-coupled redox chemistry occurs at the oxygen-evolving complex, which contains a tetramanganese cluster, a redox-active tyrosine amino acid hydrogen-bonded to a histidine amino acid, a calcium ion and chloride. Hydrogen-atom abstraction by the tyrosyl radical from water bound to the manganese cluster is now widely held to occur in this process, at least for some of the steps in the catalytic cycle. We discuss kinetic and energetic constraints on the hydrogen-atom abstraction process.

Photochemical Tyrosine Oxidation in the Structurally Well-Defined α3Y Protein: Proton-Coupled Electron Transfer and a Long-Lived Tyrosine Radical

Tyrosine oxidation–reduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The α3Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of α3Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in α3Y. Time-resolved spectroscopy using a soluble flash-quench generated [Ru(2,2′-bipyridine)3]3+ oxidant provides high-quality Y32–O• absorption spectra. The rate constant of Y32 oxidation (kPCET) is pH dependent: 1.4 × 104 M–1 s–1 (pH 5.5), 1.8 × 105 M–1 s–1 (pH 8.5), 5.4 × 103 M–1 s–1 (pD 5.5), and 4.0 × 104 M–1 s–1 (pD 8.5). kH/kD of Y32 oxidation is 2.5 ± 0.5 and 4.5 ± 0.9 at pH(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32–O• is 28–58% versus the concentration of [Ru(2,2′-bipyridine)3]3+. Y32–O• decays slowly, t1/2 in the range of 2–10 s, at both pH 5.5 and 8.5, via radical–radical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32–O• is discussed relative to the structural properties of the Y32 site. Finally, the static α3Y NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32–O• come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of α3Y.

Electrochemical and structural properties of a protein system designed to generate tyrosine Pourbaix diagrams

This report describes a model protein specifically tailored to electrochemically study the reduction potential of protein tyrosine radicals as a function of pH. The model system is based on the 67-residue α(3)Y three-helix bundle. α(3)Y contains a single buried tyrosine at position 32 and displays structural properties inherent to a protein. The present report presents differential pulse voltammograms obtained from α(3)Y at both acidic (pH 5.6) and alkaline (pH 8.3) conditions. The observed Faradaic response is uniquely associated with Y32, as shown by site-directed mutagenesis. This is the first time voltammetry is successfully applied to detect a redox-active tyrosine residing in a structured protein environment. Tyrosine is a proton-coupled electron-transfer cofactor making voltammetry-based pH titrations a central experimental approach. A second set of experiments was performed to demonstrate that pH-dependent studies can be conducted on the redox-active tyrosine without introducing large-scale structural changes in the protein scaffold. α(3)Y was re-engineered with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring. α(3)Y-K29H and α(3)Y-K36H each contain a histidine residue whose protonation perturbs the fluorescence of Y32. We show that these variants are stable and well-folded proteins whose helical content, tertiary structure, solution aggregation state, and solvent-sequestered position of Y32 remain pH insensitive across a range of at least 3-4 pH units. These results confirm that the local environment of Y32 can be altered and the resulting radical site studied by voltammetry over a broad pH range without interference from long-range structural effects.

Reversible phenol oxidation and reduction in the structurally well-defined 2-Mercaptophenol-α₃C protein.

2-Mercaptophenol-α₃C serves as a biomimetic model for enzymes that use tyrosine residues in redox catalysis and multistep electron transfer. This model protein was tailored for electrochemical studies of phenol oxidation and reduction with specific emphasis on the redox-driven protonic reactions occurring at the phenol oxygen. This protein contains a covalently modified 2-mercaptophenol-cysteine residue. The radical site and the phenol compound were specifically chosen to bury the phenol OH group inside the protein. A solution nuclear magnetic resonance structural analysis (i) demonstrates that the synthetic 2-mercaptophenol-α₃C model protein behaves structurally as a natural protein, (ii) confirms the design of the radical site, (iii) reveals that the ligated phenol forms an interhelical hydrogen bond to glutamate 13 (phenol oxygen-carboxyl oxygen distance of 3.2 ± 0.5 Å), and (iv) suggests a proton-transfer pathway from the buried phenol OH (average solvent accessible surface area of 3 ± 5%) via glutamate 13 (average solvent accessible surface area of the carboxyl oxygens of 37 ± 18%) to the bulk solvent. A square-wave voltammetry analysis of 2-mercaptophenol-α₃C further demonstrates that (v) the phenol oxidation-reduction cycle is reversible, (vi) formal phenol reduction potentials can be obtained, and (vii) the phenol-O(•) state is long-lived with an estimated lifetime of ≥180 millisecond. These properties make 2-mercaptophenol-α₃C a unique system for characterizing phenol-based proton-coupled electron transfer in a low-dielectric and structured protein environment.

Defining the Apoptotic Trigger: THE INTERACTION OF CYTOCHROME c AND CARDIOLIPIN.

Background: Binding of mitochondrial cardiolipin to cytochrome c is thought to trigger apoptosis. Results: Reverse micelle encapsulation mimics the confinement of the inner mitochrondrial membrane allowing binding of cardiolipin to be followed by NMR at atomic resolution. Conclusion: Cardiolipin interacts with cytochrome c without causing protein unfolding. Significance: A new model for the role of cytochrome c in apoptosis is required. The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (bb ∼ 0.23 Å) using NMR-based methods and is closely similar to the cryogenic crystal structure (bb ∼ 1.2 Å). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe-36, Gly-37, Thr-58, Trp-59, and Lys-60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.

Solution structure of the core SMN-Gemin2 complex.

In humans, assembly of spliceosomal snRNPs (small nuclear ribonucleoproteins) begins in the cytoplasm where the multi-protein SMN (survival of motor neuron) complex mediates the formation of a seven-membered ring of Sm proteins on to a conserved site of the snRNA (small nuclear RNA). The SMN complex contains the SMN protein Gemin2 and several additional Gemins that participate in snRNP biosynthesis. SMN was first identified as the product of a gene found to be deleted or mutated in patients with the neurodegenerative disease SMA (spinal muscular atrophy), the leading genetic cause of infant mortality. In the present study, we report the solution structure of Gemin2 bound to the Gemin2-binding domain of SMN determined by NMR spectroscopy. This complex reveals the structure of Gemin2, how Gemin2 binds to SMN and the roles of conserved SMN residues near the binding interface. Surprisingly, several conserved SMN residues, including the sites of two SMA patient mutations, are not required for binding to Gemin2. Instead, they form a conserved SMN/Gemin2 surface that may be functionally important for snRNP assembly. The SMN–Gemin2 structure explains how Gemin2 is stabilized by SMN and establishes a framework for structure–function studies to investigate snRNP biogenesis as well as biological processes involving Gemin2 that do not involve snRNP assembly.