Structural Effects of Ammonia Binding to the Mn_4CaO_5 Cluster of Photosystem II

The Mn_4CaO_5 oxygen-evolving complex (OEC) of photosystem II catalyzes the light-driven oxidation of two substrate waters to molecular oxygen. ELDOR-detected NMR along with computational studies indicated that ammonia, a substrate analogue, binds as a terminal ligand to the Mn4A ion trans to the O5 μ_4 oxido bridge. Results from electron spin echo envelope modulation (ESEEM) spectroscopy confirmed this and showed that ammonia hydrogen bonds to the carboxylate side chain of D1-Asp61. Here we further probe the environment of OEC with an emphasis on the proximity of exchangeable protons, comparing ammonia-bound and unbound forms. Our ESEEM and electron nuclear double resonance (ENDOR) results indicate that ammonia substitutes for the W1 terminal water ligand without significantly altering the electronic structure of the OEC

Biophysical Characterization of Fluorotyrosine Probes Site-Specifically Incorporated into Enzymes:

Fluorinated tyrosines (F[subscript n]Y's, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y's, a stable Y radical (Y·) in the β subunit (position 122 in E. coli), and three transiently oxidized Y's (356 in β and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. F[subscript n]Y (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y₁₂₂-β and the X-ray structures of each resulting β with a diferric cluster are reported and compared with wt-β2 crystallized under the same conditions. The essential diferric-F[subscript n]Y· cofactor is self-assembled from apo F[subscript n]Y-β2, Fe ²⁺, and O₂ to produce ∼1 Y·/β2 and ∼3 Fe ³⁺ /β2. The F[subscript n]Y· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each F[subscript n] Y·-β2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the 19 F nucleus provide unique signatures of each F[subscript n]Y· that are readily distinguishable from unlabeled Y·'s. The variability of the abiotic F[subscript n]Y pK a 's (6.4 to 7.8) and reduction potentials (-30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·'s in catalysis and to investigate the importance and identity of hopping Y·'s within redox active proteins proposed to protect them from uncoupled radical chemistry.National Institutes of Health (U.S.) (Grant GM29595)National Science Foundation (U.S.) (Grant 0645960

Role of oxido incorporation and ligand lability in expanding redox accessibility of structurally related Mn4 clusters

Photosystem II supports four manganese centers through nine oxidation states from manganese(II) during assembly through to the most oxidized state before O_2 formation and release. The protein-based carboxylate and imidazole ligands allow for significant changes of the coordination environment during the incorporation of hydroxido and oxido ligands upon oxidation of the metal centers. We report the synthesis and characterization of a series of tetramanganese complexes in four of the six oxidation states from Mn^(II)_(3)Mn^III to Mn^(III)_(2)Mn^(IV)_2 with the same ligand framework (L) by incorporating four oxido ligands. A 1,3,5-triarylbenzene framework appended with six pyridyl and three alkoxy groups was utilized along with three acetate anions to access tetramanganese complexes, Mn_(4)O_x, with x = 1, 2, 3, and 4. Alongside two previously reported complexes, four new clusters in various states were isolated and characterized by crystallography, and four were observed electrochemically, thus accessing the eight oxidation states from Mn^(II)_4 to Mn^(III)Mn^(IV)_3. This structurally related series of compounds was characterized by EXAFS, XANES, EPR, magnetism, and cyclic voltammetry. Similar to the ligands in the active site of the protein, the ancillary ligand (L) is preserved throughout the series and changes its binding mode between the low and high oxido-content clusters. Implications for the rational assembly and properties of high oxidation state metal–oxido clusters are presented

Structural Effects of Ammonia Binding to the Mn_4CaO_5 Cluster of Photosystem II

The Mn_4CaO_5 oxygen-evolving complex (OEC) of photosystem II catalyzes the light-driven oxidation of two substrate waters to molecular oxygen. ELDOR-detected NMR along with computational studies indicated that ammonia, a substrate analogue, binds as a terminal ligand to the Mn4A ion trans to the O5 μ_4 oxido bridge. Results from electron spin echo envelope modulation (ESEEM) spectroscopy confirmed this and showed that ammonia hydrogen bonds to the carboxylate side chain of D1-Asp61. Here we further probe the environment of OEC with an emphasis on the proximity of exchangeable protons, comparing ammonia-bound and unbound forms. Our ESEEM and electron nuclear double resonance (ENDOR) results indicate that ammonia substitutes for the W1 terminal water ligand without significantly altering the electronic structure of the OEC

Effects of Lewis Acidic Metal Ions (M) on Oxygen-Atom Transfer Reactivity of Heterometallic Mn_3MO_4 Cubane and Fe_3MO(OH) and Mn_3MO(OH) Clusters

The modulation of the reactivity of metal oxo species by redox inactive metals has attracted much interest due to the observation of redox inactive metal effects on processes involving electron transfer both in nature (the oxygen-evolving complex of Photosystem II) and in heterogeneous catalysis (mixed-metal oxides). Studies of small-molecule models of these systems have revealed numerous instances of effects of redox inactive metals on electron- and group-transfer reactivity. However, the heterometallic species directly involved in these transformations have rarely been structurally characterized and are often generated in situ. We have previously reported the preparation and structural characterization of multiple series of heterometallic clusters based on Mn_3 and Fe_3 cores and described the effects of Lewis acidity of the heterometal incorporated in these complexes on cluster reduction potential. To determine the effects of Lewis acidity of redox inactive metals on group transfer reactivity in structurally well-defined complexes, we studied [Mn_3MO_4], [Mn_3MO(OH)], and [Fe_3MO(OH)] clusters in oxygen atom transfer (OAT) reactions with phosphine substrates. The qualitative rate of OAT correlates with the Lewis acidity of the redox inactive metal, confirming that Lewis acidic metal centers can affect the chemical reactivity of metal oxo species by modulating cluster electronics

Redox, haem and CO in enzymatic catalysis and regulation

The present paper describes general principles of redox catalysis and redox regulation in two diverse systems. The first is microbial metabolism of CO by the Wood–Ljungdahl pathway, which involves the conversion of CO or H2/CO2 into acetyl-CoA, which then serves as a source of ATP and cell carbon. The focus is on two enzymes that make and utilize CO, CODH (carbon monoxide dehydrogenase) and ACS (acetyl-CoA synthase). In this pathway, CODH converts CO2 into CO and ACS generates acetyl-CoA in a reaction involving Ni·CO, methyl-Ni and acetyl-Ni as catalytic intermediates. A 70 Å (1 Å=0.1 nm) channel guides CO, generated at the active site of CODH, to a CO ‘cage’ near the ACS active site to sequester this reactive species and assure its rapid availability to participate in a kinetically coupled reaction with an unstable Ni(I) state that was recently trapped by photolytic, rapid kinetic and spectroscopic studies. The present paper also describes studies of two haem-regulated systems that involve a principle of metabolic regulation interlinking redox, haem and CO. Recent studies with HO2 (haem oxygenase-2), a K+ ion channel (the BK channel) and a nuclear receptor (Rev-Erb) demonstrate that this mode of regulation involves a thiol–disulfide redox switch that regulates haem binding and that gas signalling molecules (CO and NO) modulate the effect of haem.National Institutes of Health (U.S.) (NIH grant GM69857)National Institutes of Health (U.S.) (NIH grant GM39451)National Institutes of Health (U.S.) (NIH grant HL 102662)National Institutes of Health (U.S.) (NIH grant GM65440)National Institutes of Health (U.S.) (NIH grant GM48242)National Institutes of Health (U.S.) (NIH grant Y1-GM- 1104)National Institutes of Health (U.S.) (NIH grant GM065318)National Institutes of Health (U.S.) (NIH grant AG027349)National Science Foundation (U.S.) (grant number CHE-0745353)United States. Dept. of Energy. Office of Biological and Environmental ResearchHoward Hughes Medical Institute (Investigator

Mechanism of Assembly of the Dimanganese-Tyrosyl Radical Cofactor of Class Ib Ribonucleotide Reductase: Enzymatic Generation of Superoxide Is Required for Tyrosine Oxidation via a Mn(III)Mn(IV) Intermediate

Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The Fe[superscript III][subscript 2]-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe[superscript II][subscript 2]-NrdB, O[subscript 2], and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn[superscript III][subscript 2]-Y• cofactor in their NrdF subunit. Mn[superscript II][subscript 2]-NrdF does not react with O[subscript 2], but it binds the reduced form of a conserved flavodoxin-like protein, NrdI[subscript hq], which, in the presence of O[subscript 2], reacts to form the Mn[superscript III][subscript 2]-Y• cofactor. Here we investigate the mechanism of assembly of the Mn[superscript III][subscript 2]-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn[superscript II][subscript 2]-NrdF, NrdI[subscript hq], and O[subscript 2] has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI[subscript hq] reduces O[subscript 2] to O[subscript 2]•– (40–48 s[superscript –1], 0.6 mM O[subscript 2]), the O[subscript 2]•– channels to and reacts with Mn[superscript II][subscript 2]-NrdF to form a Mn[superscript III]Mn[superscript IV] intermediate (2.2 ± 0.4 s[superscript –1]), and the Mn[superscript III]Mn[superscript IV] species oxidizes tyrosine to Y• (0.08–0.15 s[superscript –1]). Controlled production of O[subscript 2]•– by NrdI[subscript hq] during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn[superscript II][subscript 2] cluster with O[subscript 2] and satisfies the requirement for an “extra” reducing equivalent in Y• generation.National Institutes of Health (U.S.) (Grant GM81393)United States. Dept. of Defense (National Defense Science and Engineering Graduate (NDSEG) Fellowships

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)