Post-compilation optimization for multiple gains with pattern matching

Many existing retargetable compilers for ASIPs and domain-specific processors generate low quality code since the compiler is not able to fully utilize the intricacies of ISA of these processors. Hence, there is a need to further optimize the code produced by these compilers. In this paper, we introduce a new post-compilation optimization technique which is based on finding repeating instruction patterns in generated code and replacing them with their optimized equivalents. The instruction patterns to be found are represented by finite state machines which allow encapsulation of multiple patterns in just one representation, and instructions in a pattern to be not necessarily lexically adjacent. We also present a conflict resolution algorithm to select an optimization whenever a set of instructions fall under two or more different patterns of which only one can be applied on the basis of code size, cycle count or switching activity improvement. We tested this technique on the compiled binaries of ARM and Intel processors for code size improvement. We discuss the possible applications of this strategy in design space exploration (DSE) of embedded processors

The catalytic mechanism for aerobic formation of methane by bacteria. Nature 497, 132–136. doi: 10.1038/nature12061

Methane is a potent greenhouse gas that is produced in significant quantities by aerobic marine organisms 1 . These bacteria apparently catalyse the formation of methane through the cleavage of the highly unreactive carbon-phosphorus bond in methyl phosphonate (MPn), but the biological or terrestrial source of this compound is unclear 2 . However, the ocean-dwelling bacterium Nitrosopumilus maritimus catalyses the biosynthesis of MPn from 2-hydroxyethyl phosphonate 3 and the bacterial C-P lyase complex is known to convert MPn to methane Here we show that PhnJ is a novel radical S-adenosyl-L-methionine enzyme that catalyses C-P bond cleavage through the initial formation of a 59-deoxyadenosyl radical and two protein-based radicals localized at Gly 32 and Cys 272. During this transformation, the pro-R hydrogen from Gly 32 is transferred to the 59-deoxyadenosyl radical to form 59-deoxyadenosine and the pro-S hydrogen is transferred to the radical intermediate that ultimately generates methane. A comprehensive reaction mechanism is proposed for cleavage of the C-P bond by the C-P lyase complex that uses a covalent thiophosphate intermediate for methane and phosphate formation. The glutathione S-transferase (GST) fusion protein of PhnJ from Escherichia coli was purified under anaerobic conditions 8 . The isolated protein was dark brown in colour, had an absorbance maximum at a wavelength of 410 nm and was EPR silent (produced no electron paramagnetic resonance signal) It was shown previously that 59-deoxyadenosine (Ado-CH 3 ) and L-methionine are formed from the utilization of SAM during the reaction catalysed by PhnJ and that approximately one enzyme equivalent of SAM is consumed under single or multiple turnover

Characterization of a High-Spin Non-Heme Fe-III-O Intermediate and Its Quantitative Conversion to an Fe-IV = O Complex

We have generated a high-spin Fe-III-OOH complex supported by tetramethylcyclam via protonation of its conjugate base and characterized it in detail using various spectroscopic methods. This Fe-III-OOH species can be converted quantitatively to an Fe-IV=O complex via O-O bond cleavage; this is the first example of such a conversion. This conversion is promoted by two factors: the strong Fe-III-OOH bond, which inhibits Fe-O bond lysis, and the addition of protons, which facilitates O-O bond cleavage. This example provides a synthetic precedent for how O-O bond cleavage of high-spin Fe-III-peroxo intermediates of non-heme iron enzymes may be promoted

Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe–S Assembly Complex

Iron–sulfur (Fe–S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe–S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe–S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. <i>In vitro</i> assays have relied on reducing the complexity of this complicated Fe–S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe–S assembly complex. Here the kinetics of Fe–S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe–S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) <i>Biochemistry 54</i>, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe–S assembly complex

High-Spin Ferric Ions in Saccharomyces cerevisiae Vacuoles Are Reduced to the Ferrous State during Adenine-Precursor Detoxification

The majority of Fe in Fe-replete yeast cells is located in vacuoles. These acidic organelles store Fe for use under Fe-deficient conditions and they sequester it from other parts of the cell to avoid Fe-associated toxicity. Vacuolar Fe is predominantly in the form of one or more magnetically isolated nonheme high-spin (NHHS) Fe^III complexes with polyphosphate-related ligands. Some Fe^III oxyhydroxide nanoparticles may also be present in these organelles, perhaps in equilibrium with the NHHS Fe^III. Little is known regarding the chemical properties of vacuolar Fe. When grown on adenine-deficient medium (A↓), ADE2Δ strains of yeast such as W303 produce a toxic intermediate in the adenine biosynthetic pathway. This intermediate is conjugated with glutathione and shuttled into the vacuole for detoxification. The iron content of A↓ W303 cells was determined by Mössbauer and EPR spectroscopies. As they transitioned from exponential growth to stationary state, A↓ cells (supplemented with 40 μM Fe^III citrate) accumulated two major NHHS Fe^II species as the vacuolar NHHS Fe^III species declined. This is evidence that vacuoles in A↓ cells are more reducing than those in adenine-sufficient cells. A↓ cells suffered less oxidative stress despite the abundance of NHHS Fe^II complexes; such species typically promote Fenton chemistry. Most Fe in cells grown for 5 days with extra yeast-nitrogen-base, amino acids and bases in minimal medium was HS Fe^III with insignificant amounts of nanoparticles. The vacuoles of these cells might be more acidic than normal and can accommodate high concentrations of HS Fe^III species. Glucose levels and rapamycin (affecting the TOR system) affected cellular Fe content. This study illustrates the sensitivity of cellular Fe to changes in metabolism, redox state and pH. Such effects broaden our understanding of how Fe and overall cellular metabolism are integrated

Mössbauer, EPR, and Modeling Study of Iron Trafficking and Regulation in <i>Δccc1</i> and <i>CCC1-up Saccharomyces cerevisiae</i>

Strains lacking and overexpressing the vacuolar iron (Fe) importer <i>CCC1</i> were characterized using Mössbauer and EPR spectroscopies. Vacuolar Fe import is impeded in <i>Δccc1</i> cells and enhanced in <i>CCC1-up</i> cells, causing vacuolar Fe in these strains to decline and accumulate, respectively, relative to WT cells. Cytosolic Fe levels should behave oppositely. The Fe content of <i>Δccc1</i> cells grown under low-Fe conditions was similar to that in WT cells. Most Fe was mitochondrial with some nonheme high spin (NHHS) Fe^II present. <i>Δccc1</i> cells grown with increasing Fe concentration in the medium contained less total Fe, less vacuolar HS Fe^III, and more NHHS Fe^II than in comparable WT cells. As the Fe concentration in the growth medium increased, the concentration of HS Fe^III in <i>Δccc1</i> cells increased to just 60% of WT levels, while NHHS Fe^II increased to twice WT levels, suggesting that the NHHS Fe^II was cytosolic. <i>Δccc1</i> cells suffered more oxidative damage than WT cells, suggesting that the accumulated NHHS Fe^II promoted Fenton chemistry. The Fe concentration in <i>CCC1-up</i> cells was higher than in WT cells; the extra Fe was present as NHHS Fe^II and Fe^III and as Fe^III oxyhydroxide nanoparticles. These cells contained less mitochondrial Fe and exhibited less ROS damage than <i>Δccc1</i> cells. <i>CCC1-up</i> cells were adenine-deficient on minimal medium; supplementing with adenine caused a decline of NHHS Fe^II suggesting that some of the NHHS Fe^II that accumulated in these cells was associated with adenine deficiency rather than the overexpression of <i>CCC1</i>. A mathematical model was developed that simulated changes in Fe distributions. Simulations suggested that only a modest proportion of the observed NHHS Fe^II in both strains was the cytosolic form of Fe that is sensed by the Fe import regulatory system. The remainder is probably generated by the reduction of the vacuolar NHHS Fe^III species

Mössbauer Study and Modeling of Iron Import and Trafficking in Human Jurkat Cells

The Fe content of Jurkat cells grown on transferrin-bound iron (TBI) and Fe^III citrate (FC) was characterized using Mössbauer, electron paramagnetic resonance, and UV–vis spectroscopies, as well as electron and inductively coupled plasma mass spectrometry. Isolated mitochondria were similarly characterized. Fe-limited cells contained ∼100 μM essential Fe, mainly as mitochondrial Fe and nonmitochondrial non-heme high-spin Fe^II. Cells replete with Fe also contained ferritin-bound Fe and Fe^III oxyhydroxide nanoparticles. Only 400 ± 100 Fe ions were loaded per ferritin complex, regardless of the growth medium Fe concentration. Ferritin regulation thus appears to be more complex than is commonly assumed. The magnetic and structural properties of Jurkat nanoparticles differed from those of yeast mitochondria. They were smaller and may be located in the cytosol. The extent of nanoparticle formation scaled nonlinearly with the concentration of Fe in the medium. Nanoparticle formation was not strongly correlated with reactive oxygen species (ROS) damage. Cells could utilize nanoparticle Fe, converting such aggregates into essential Fe forms. Cells grown on galactose rather than glucose respired faster, grew slower, exhibited more ROS damage, and generally contained more nanoparticles. Cells grown with TBI rather than FC contained less Fe overall, more ferritin, and fewer nanoparticles. Cells in which the level of transferrin receptor expression was increased contained more ferritin Fe. Frataxin-deficient cells contained more nanoparticles than comparable wild-type cells. Data were analyzed by a chemically based mathematical model. Although simple, it captured essential features of Fe import, trafficking, and regulation. TBI import was highly regulated, but FC import was not. Nanoparticle formation was not regulated, but the rate was third-order in cytosolic Fe

Biophysical Investigation of the Ironome of Human Jurkat Cells and Mitochondria

The speciation of iron in intact human Jurkat leukemic cells and their isolated mitochondria was assessed using biophysical methods. Large-scale cultures were grown in medium enriched with ⁵⁷Fe citrate. Mitochondria were isolated anaerobically to prevent oxidation of iron centers. 5 K Mössbauer spectra of cells were dominated by a sextet due to ferritin. They also exhibited an intense central quadrupole doublet due to <i>S</i> = 0 [Fe₄S₄]²⁺ clusters and low-spin (LS) Fe^II heme centers. Spectra of isolated mitochondria were largely devoid of ferritin but contained the central doublet and features arising from what appear to be Fe^III oxyhydroxide (phosphate) nanoparticles. Spectra from both cells and mitochondria contained a low-intensity doublet from non-heme high-spin (NHHS) Fe^II species. A portion of these species may constitute the “labile iron pool” (LIP) proposed in cellular Fe trafficking. Such species might engage in Fenton chemistry to generate reactive oxygen species. Electron paramagnetic resonance spectra of cells and mitochondria exhibited signals from reduced Fe/S clusters, and HS Fe^III heme and non-heme species. The basal heme redox state of mitochondria within cells was reduced; this redox poise was unaltered during the anaerobic isolation of the organelle. Contributions from heme <i>a</i>, <i>b</i>, and <i>c</i> centers were quantified using electronic absorption spectroscopy. Metal concentrations in cells and mitochondria were measured using inductively coupled plasma mass spectrometry. Results were collectively assessed to estimate the concentrations of various Fe-containing species in mitochondria and whole cells  the first “ironome” profile of a human cell

Mixed-Valence Nickel–Iron Dithiolate Models of the [NiFe]-Hydrogenase Active Site

A series of mixed-valence nickel–iron dithiolates is described. Oxidation of (diphosphine)­Ni­(dithiolate)­Fe­(CO)₃ complexes <b>1</b>, <b>2</b>, and <b>3</b> with ferrocenium salts affords the corresponding tricarbonyl cations [(dppe)­Ni­(pdt)­Fe­(CO)₃]⁺ ([<b>1</b>]⁺), [(dppe)­Ni­(edt)­Fe­(CO)₃]⁺ ([<b>2</b>]⁺) and [(dcpe)­Ni­(pdt)­Fe­(CO)₃]⁺ ([<b>3</b>]⁺), respectively, where dppe = Ph₂PCH₂CH₂PPh₂, dcpe = Cy₂PCH₂CH₂PCy₂, (Cy = cyclohexyl), pdtH₂ = HSCH₂CH₂CH₂SH, and edtH₂ = HSCH₂CH₂SH. The cation [<b>2</b>]⁺ proved unstable, but the propanedithiolates are robust. IR and EPR spectroscopic measurements indicate that these species exist as <i>C</i>_<i>s</i>-symmetric species. Crystallographic characterization of [<b>3</b>]­BF₄ shows that Ni is square planar. Interaction of [<b>1</b>]­BF₄ with P-donor ligands (L) afforded a series of substituted derivatives of type [(dppe)­Ni­(pdt)­Fe­(CO)₂L]­BF₄ for L = P­(OPh)₃ ([<b>4a</b>]­BF₄), P­(<i>p</i>-C₆H₄Cl)₃ ([<b>4b</b>]­BF₄), PPh₂(2-py) ([<b>4c</b>]­BF₄), PPh₂(OEt) ([<b>4d</b>]­BF₄), PPh₃ ([<b>4e</b>]­BF₄), PPh₂(<i>o</i>-C₆H₄OMe) ([<b>4f</b>]­BF₄), PPh₂(<i>o</i>-C₆H₄OCH₂OMe) ([<b>4g</b>]­BF₄), P­(<i>p</i>-tol)₃ ([<b>4h</b>]­BF₄), P­(<i>p</i>-C₆H₄OMe)₃ ([<b>4i</b>]­BF₄), and PMePh₂ ([<b>4j</b>]­BF₄). EPR analysis indicates that ethanedithiolate [<b>2</b>]⁺ exists as a single species at 110 K, whereas the propanedithiolate cations exist as a mixture of two conformers, which are proposed to be related through a flip of the chelate ring. Mössbauer spectra of <b>1</b> and oxidized <i>S</i> = 1/2 [<b>4e</b>]­BF₄ are both consistent with a low-spin Fe­(I) state. The hyperfine coupling tensor of [<b>4e</b>]­BF₄ has a small isotropic component and significant anisotropy. DFT calculations using the BP86, B3LYP, and PBE0 exchange–correlation functionals agree with the structural and spectroscopic data, suggesting that the SOMOs in complexes of the present type are localized in an Fe­(I)-centered d­(<i>z</i>²) orbital. The DFT calculations allow an assignment of oxidation states of the metals and rationalization of the conformers detected by EPR spectroscopy. Treatment of [<b>1</b>]⁺ with CN^– and compact basic phosphines results in complex reactions. With dppe, [<b>1</b>]⁺ undergoes quasi-disproportionation to give <b>1</b> and the diamagnetic complex [(dppe)­Ni­(pdt)­Fe­(CO)₂(dppe)]²⁺ ([<b>5</b>]²⁺), which features square-planar Ni linked to an octahedral Fe center

Mössbauer and EPR Study of Iron in Vacuoles from Fermenting <i>Saccharomyces cerevisiae</i>

Vacuoles were isolated from fermenting yeast cells grown on minimal medium supplemented with 40 μM ⁵⁷Fe. Absolute concentrations of Fe, Cu, Zn, Mn, Ca, and P in isolated vacuoles were determined by ICP-MS. Mössbauer spectra of isolated vacuoles were dominated by two spectral features: a mononuclear magnetically isolated high-spin (HS) Fe^III species coordinated primarily by hard/ionic (mostly or exclusively oxygen) ligands and superparamagnetic Fe^III oxyhydroxo nanoparticles. EPR spectra of isolated vacuoles exhibited a <i>g</i>_ave ∼ 4.3 signal typical of HS Fe^III with E/D ∼ 1/3. Chemical reduction of the HS Fe^III species was possible, affording a Mössbauer quadrupole doublet with parameters consistent with O/N ligation. Vacuolar spectral features were present in whole fermenting yeast cells; however, quantitative comparisons indicated that Fe leaches out of vacuoles during isolation. The <i>in vivo</i> vacuolar Fe concentration was estimated to be ∼1.2 mM while the Fe concentration of isolated vacuoles was ∼220 μM. Mössbauer analysis of Fe^III polyphosphate exhibited properties similar to those of vacuolar Fe. At the vacuolar pH of 5, Fe^III polyphosphate was magnetically isolated, while at pH 7, it formed nanoparticles. This pH-dependent conversion was reversible. Fe^III polyphosphate could also be reduced to the Fe^II state, affording similar Mössbauer parameters to that of reduced vacuolar Fe. These results are insufficient to identify the exact coordination environment of the Fe^III species in vacuoles, but they suggest a complex closely related to Fe^III polyphosphate. A model for Fe trafficking into/out of yeast vacuoles is proposed