Jeffrey M. Peloquin

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).

Probing the coupling between proton and electron transfer in photosystem II core complexes containing a 3-fluorotyrosine.

The catalytic cycle of numerous enzymes involves the coupling between proton transfer and electron transfer. Yet, the understanding of this coordinated transfer in biological systems remains limited, likely because its characterization relies on the controlled but experimentally challenging modifications of the free energy changes associated with either the electron or proton transfer. We have performed such a study here in Photosystem II. The driving force for electron transfer from Tyr(Z) to P(680)(*+) has been decreased by approximately 80 meV by mutating the axial ligand of P(680), and that for proton transfer upon oxidation of Tyr(Z) by substituting a 3-fluorotyrosine (3F-Tyr(Z)) for Tyr(Z). In Mn-depleted Photosystem II, the dependence upon pH of the oxidation rates of Tyr(Z) and 3F-Tyr(Z) were found to be similar. However, in the pH range where the phenolic hydroxyl of Tyr(Z) is involved in a H-bond with a proton acceptor, the activation energy of the oxidation of 3F-Tyr(Z) is decreased by 110 meV, a value which correlates with the in vitro finding of a 90 meV stabilization energy to the phenolate form of 3F-Tyr when compared to Tyr (Seyedsayamdost et al. J. Am. Chem. Soc. 2006, 128,1569-1579). Thus, when the phenol of Y(Z) acts as a H-bond donor, its oxidation by P(680)(*+) is controlled by its prior deprotonation. This contrasts with the situation prevailing at lower pH, where the proton acceptor is protonated and therefore unavailable, in which the oxidation-induced proton transfer from the phenolic hydroxyl of Tyr(Z) has been proposed to occur concertedly with the electron transfer to P(680)(*+). This suggests a switch between a concerted proton/electron transfer at pHs < 7.5 to a sequential one at pHs > 7.5 and illustrates the roles of the H-bond and of the likely salt-bridge existing between the phenolate and the nearby proton acceptor in determining the coupling between proton and electron transfer.

The role of reaction center excited state evolution during charge separation in a Rb. sphaeroides mutant with an initial electron donor midpoint potential 260 mV above wild type

Abstract Femtosecond transient absorbance spectroscopy was performed on the triple hydrogen bond reaction center mutant [LH(L131) + LH(M160) + FH(M197)] of Rhodobacter sphaeroides which has a P/P+ midpoint potential 260 mV above wild type. The decay of the excited singlet state in this mutant is kinetically complex with a dominant decay component of about 50 ps at 295 K. Charge separation to the state P+QA− occurs with a quantum yield of 0.50 ± 0.1 at 295 K and 0.10–0.15 at 20 K. The yield, rate of formation and spectra of states which are trapped when electron transfer to the quinone is blocked by quinone reduction compared to the rate and yield of formation of P+QA− in unreduced reaction centers suggest that evolution of the excited state is the rate limiting event in charge separation in triple mutant reaction centers. The excited state that results from this evolution has spectral features which are remarkably similar to the initial excited singlet state found using R-26 reaction centers (R-26 reaction centers have essentially wild type photochemistry). The fact that the formation of this altered excited state is greatly slowed in a high P/P+ midpoint potential mutant suggests that the early excited state in wild type or R-26 reaction centers may have considerable P+ character. A consideration of the thermodynamics of the state P+BA− in this and related high potential mutants implies that a simple model in which P+BA− is formed as a discrete electron transfer intermediate is not a viable description in these mutants. Other factors such as reaction center heterogeneity or alternate electron transfer mechanisms must be invoked.

Reduction of 13-deoxydoxorubicin and daunorubicinol anthraquinones by human carbonyl reductase.

Carbonyl reductase (CR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of several carbonyls. Anthracyclines used to treat cancer are reduced by CR at the C13 carbonyl and the resulting metabolites are implicated in the cardiotoxicity associated with anthracycline therapy. CR also is believed to have a role in detoxifying quinones, raising the question whether CR catalyzes reduction of anthracycline quinones. Steadystate kinetic studies were done with several anthraquinone-containing compounds, including 13-deoxydoxorubicin and daunorubicinol, which lack the C13 carbonyl, thus unmasking the anthraquinone for study. kcat and kcat/Km values for 13-deoxydoxorubicin and daunorubicinol were nearly identical, indicating that that the efficiency of quinone reduction was unaffected by the differences at the C13 position. kcat and kcat/Km values were much smaller for the analogs than for the parent compounds, suggesting that the C13 carbonyl is preferred as a substrate over the quinone. CR also reduced structurally related quinone molecules with less favorable catalytic efficiency. Modeling studies with doxorubicin and carbonyl reductase revealed that methionine 234 sterically hinder the rings adjacent to the quinone, thus accounting for the lower catalytic efficiency. Reduction of the anthraquinones may further define the role of CR in anthracycline metabolism and may influence anthracycline cytotoxic and cardiotoxic mechanisms.

ESEEM studies of succinate:ubiquinone reductase from Paracoccus denitrificans.

Abstract. Electron spin-echo envelope modulation (ESEEM) spectroscopy has been performed in order to obtain structural information about the environment of the reduced [2Fe-2S] cluster (S-1 center), the oxidized [3Fe-4S] cluster (S-3 center), and the flavin semiquinone radical in purified succinate:ubiquinone reductase from Paracoccus denitrificans. Spectral simulations of the ESEEM data from the reduced [2Fe-2S] yielded nuclear quadrupole interaction parameters that are indicative of peptide nitrogens. We also observed a weak interaction between the oxidized [3Fe-4S] cluster and a peptide 14N. There was no evidence for coordination of any of the Fe atoms to 14N atoms of imidazole rings. The ESEEM data from the flavin semiquinone radical were more complicated. Here, evidence was obtained for interactions between the unpaired electron and only the two nitrogen atoms in the flavin ring.

Characterization of Sn, Zn, In, and Sb-Containing GeSe Alloys for Phase-Change Electronic Memory Applications

Two-terminal electronic devices consisting of stacks of chalcogenide layers containing GeTe, Ge 40 Se 60 , SnTe, or SnSe have shown promise for application as electronic phase-change memories (Campbell, K.A. and Anderson, C.M., Microelectronics Journal 38, 52–59 (2007)). Here, we report the synthesis of (Ge 40 Se 60 ) 100−z Mz alloys where M = Sn, In, Sb, and Zn, and the corresponding bulk material Raman spectra and differential scanning calorimetry data in order to explore material compositions that might be useful for producing a multi-state phase-change memory device.

Mutations that Affect the Donor Midpoint Potential in Reaction Centers from Rhodobacter Sphaeroides

Modulation of the redox midpoint potential of the initial electron donor is key for achieving electron transfer with high yields in photosynthetic systems. The initial electron donor in reaction centers of purple nonsulfur bacteria, P, has a much lower potential than the donor in photosystem II reaction centers, which are capable of oxidizing water (for general reviews of chlorophylls and their oxidation potentials, see ref. 1). Isolated bacteriochlorophyll and chlorophyll have similar oxidation potentials of approximately 700 mV and 800 mV, respectively. However, in vivo the midpoint oxidation potential of the donor in bacterial systems, approximately 490 mV, is much lower than that of monomer bacteriochlorophyll, while that of the donor in photosystem II is higher than 1.0 V. This dramatic alteration of the oxidation potentials in the reaction center must be due to interactions between chlorophylls or between the donor and the surrounding protein. This is supported by the observation of changes of approximately 100 mV in the oxidation potentials for bacteriochlorophylls and chlorophylls due to changes in solvent1. In this paper we address the role of specific protein-donor interactions in modulating the potential.