Molecular understanding of heteronuclear active sites in heme-copper oxidases, nitric oxide reductases, and sulfite reductases through biomimetic modelling.

Heme-copper oxidases (HCO), nitric oxide reductases (NOR), and sulfite reductases (SiR) catalyze the multi-electron and multi-proton reductions of O2, NO, and SO32-, respectively. Each of these reactions is important to drive cellular energy production through respiratory metabolism and HCO, NOR, and SiR evolved to contain heteronuclear active sites containing heme/copper, heme/nonheme iron, and heme-[4Fe-4S] centers, respectively. The complexity of the structures and reactions of these native enzymes, along with their large sizes and/or membrane associations, make it challenging to fully understand the crucial structural features responsible for the catalytic properties of these active sites. In this review, we summarize progress that has been made to better understand these heteronuclear metalloenzymes at the molecular level though study of the native enzymes along with insights gained from biomimetic models comprising either small molecules or proteins. Further understanding the reaction selectivity of these enzymes is discussed through comparisons of their similar heteronuclear active sites, and we offer outlook for further investigations.

A Terminal FeIII-Oxo in a Tetranuclear Cluster: Effects of Distal Metal Centers on Structure and Reactivity.

Tetranuclear Fe clusters have been synthesized bearing a terminal FeIII-oxo center stabilized by hydrogen-bonding interactions from pendant ( tert-butylamino)pyrazolate ligands. This motif was supported in multiple Fe oxidation states, ranging from [FeII2FeIII2] to [FeIII4]; two oxidation states were structurally characterized by single-crystal X-ray diffraction. The reactivity of the FeIII-oxo center in proton-coupled electron transfer with X-H (X = C, O) bonds of various strengths was studied in conjunction with analysis of thermodynamic square schemes of the cluster oxidation states. These results demonstrate the important role of distal metal centers in modulating the reactivity of a terminal metal-oxo.

Zirconium complexes supported by a ferrocene-based ligand as redox switches for hydroamination reactions.

The synthesis of (thiolfan*)Zr(NEt2)2 (thiolfan* = 1,1'-bis(2,4-di-tert-butyl-6-thiophenoxy)ferrocene) and its catalytic activity for intramolecular hydroamination are reported. In situ oxidation and reduction of the metal complex results in reactivity towards different substrates. The reduced form of (thiolfan*)Zr(NEt2)2 catalyzes hydroamination reactions of primary aminoalkenes, whereas the oxidized form catalyzes hydroamination reactions of secondary aminoalkenes.

Thermodynamics of Proton and Electron Transfer in Tetranuclear Clusters with Mn-OH2/OH Motifs Relevant to H2O Activation by the Oxygen Evolving Complex in Photosystem II.

We report the synthesis of site-differentiated heterometallic clusters with three Fe centers and a single Mn site that binds water and hydroxide in multiple cluster oxidation states. Deprotonation of FeIII/II3MnII-OH2 clusters leads to internal reorganization resulting in formal oxidation at Mn to generate FeIII/II3MnIII-OH. 57Fe Mössbauer spectroscopy reveals that oxidation state changes (three for FeIII/II3Mn-OH2 and four for FeIII/II3Mn-OH clusters) occur exclusively at the Fe centers; the Mn center is formally MnII when water is bound and MnIII when hydroxide is bound. Experimentally determined p Ka (17.4) of the [FeIII2FeIIMnII-OH2] cluster and the reduction potentials of the [Fe3Mn-OH2] and [Fe3Mn-OH] clusters were used to analyze the O-H bond dissociation enthalpies (BDEO-H) for multiple cluster oxidation states. BDEO-H increases from 69 to 78 and 85 kcal/mol for the [FeIIIFeII2MnII-OH2], [FeIII2FeIIMnII-OH2], and [FeIII3MnII-OH2] clusters, respectively. Further insight of the proton and electron transfer thermodynamics of the [Fe3Mn-OH x] system was obtained by constructing a potential-p Ka diagram; the shift in reduction potentials of the [Fe3Mn-OH x] clusters in the presence of different bases supports the BDEO-H values reported for the [Fe3Mn-OH2] clusters. A lower limit of the p Ka for the hydroxide ligand of the [Fe3Mn-OH] clusters was estimated for two oxidation states. These data suggest BDEO-H values for the [FeIII2FeIIMnIII-OH] and [FeIII3MnIII-OH] clusters are greater than 93 and 103 kcal/mol, which hints to the high reactivity expected of the resulting [Fe3Mn═O] in this and related multinuclear systems.

Tetranuclear Fe Clusters with a Varied Interstitial Ligand: Effects on the Structure, Redox Properties, and Nitric Oxide Activation.

A new series of tetranuclear Fe clusters displaying an interstitial µ4-F ligand was prepared for a comparison to previously reported µ4-O analogues. With a single nitric oxide (NO) coordinated as a reporter of small-molecule activation, the µ4-F clusters were characterized in five redox states, from FeII3{FeNO}8 to FeIII3{FeNO}7, with NO stretching frequencies ranging from 1680 to 1855 cm-1, respectively. Despite accessing more reduced states with an F- bridge, a two-electron reduction of the distal Fe centers is necessary for the µ4-F clusters to activate NO to the same degree as the µ4-O system; consequently, NO reactivity is observed at more positive potentials with µ4-O than µ4-F. Moreover, the µ4-O ligand better translates redox changes of remote metal centers to diatomic ligand activation. The implication for biological active sites is that the higher-charge bridging ligand is more effective in tuning cluster properties, including the involvement of remote metal centers, for small-molecule activation.

Circular dichroism and fluorescence spectroscopy of cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 demonstrates that group I cations are particularly effective in providing structure and stability to this halophilic protein.

Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 (NRC-1), which was used as a model halophilic protein. The effects of salt on the structure and stability of NRC-1 and of E. coli CysRS were investigated through far-UV circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and thermal denaturation melts. The CD of NRC-1 CysRS was examined in different group I and group II chloride salts to examine the effects of the metal ions. Potassium was observed to have the strongest effect on NRC-1 CysRS structure, with the other group I salts having reduced strength. The group II salts had little effect on the protein. This suggests that the halophilic adaptations in this protein are mediated by potassium. CD and fluorescence spectra showed structural changes taking place in NRC-1 CysRS over the concentration range of 0-3 M KCl, while the structure of E. coli CysRS was relatively unaffected. Salt was also shown to increase the thermal stability of NRC-1 CysRS since the melt temperature of the CysRS from NRC-1 was increased in the presence of high salt, whereas the E. coli enzyme showed a decrease. By characterizing these interactions, this study not only explains the stability of halophilic proteins in extremes of salt, but also helps us to understand why and how group I salts stabilize proteins in general.

Protein adaptations in archaeal extremophiles.

Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity.

Ultrasound settings significantly alter arterial lumen and wall thickness measurements.

BACKGROUND: Flow-mediated dilation (FMD) and carotid intima-medial thickness (CIMT), measured by ultrasound, are widely used to test the efficacy of cardioprotective interventions. Although assessment methods vary, automated edge-detecting image analysis software is routinely used to measure changes in FMD and CIMT. We aimed to quantify the effect that commonly adjusted ultrasound settings have on arterial lumen and wall thickness measurements made with CIMT measurement software. METHODS: We constructed phantom arteries from a tissue-mimicking agar compound and scanned them in a water bath with a 10 MHz multi-frequency linear-array probe attached to a high-resolution ultrasound machine. B-mode images of the phantoms were recorded with dynamic range (DR) and gain set at five decibel (dB) increments from 40 dB to 60 dB and -10 dB to +10 dB respectively. Lumen diameter and wall-thickness were measured off-line using CIMT measurement software. RESULTS: Lumen measurements: there was a strong linear relationship between DR and gain and measured lumen diameter. For a given gain level, a 5 dB increase in DR reduced the measured lumen diameter by 0.02 +/- 0.004 mm (p < 0.001). For a given DR level, a 5 dB increase in gain reduced measured lumen diameter by 0.04 +/- 0.004 mm (p < 0.001). A 5 mm increase in distance between the ultrasound probe and the artery reduced measured lumen diameter by 0.04 +/- 0.03 mm (p < 0.001)CIMT measurements: For a fixed gain level, a 5 dB increase in DR increased measured wall thickness by 0.003 +/- 0.002 mm (p < 0.001). The effects of increasing gain were not consistent and appeared to vary depending on the distance between the artery and the ultrasound probe and the thickness of the artery wall. CONCLUSION: DR, gain and probe distance significantly alter lumen diameter and CIMT measurements made using image analysis software. When CIMT and FMD are used to test the efficacy of cardioprotective interventions, the DR, gain and probe position used to record baseline scans should be documented and replicated in post-treatment scans in individual trial subjects. If more than one sonographer or imaging centre is used to collect data, the study protocol should document specific DR and gain settings to be used in all subjects.

Carotid intima-medial thickness measured on multiple ultrasound frames: evaluation of a DICOM-based software system.

BACKGROUND: Carotid intima-media thickness (CIMT) measured by B-mode ultrasonography is a marker of atherosclerosis and is commonly used as an outcome in intervention trials. We have developed DICOM-based software that measures CIMT rapidly on multiple end-diastolic image frames. The aims of this study were to compare the performance of our new software with older bitmap-based CIMT measurement software and to determine whether a ten-fold increase in the number of measurements used to calculate mean CIMT would improve reproducibility. METHODS: Two independent sonographers recorded replicate carotid scans in thirty volunteers and two blinded observers measured CIMT off-line using the new DICOM-based software and older bitmap-based software. A Bland-Altman plot was used to compare CIMT results from the two software programs and t-tests were used to compare analysis times. F-tests were used to compare the co-efficients of variation (CVs) from a standard six-frame measurement protocol with CVs from a sixty-frame measurement protocol. Ordinary least products (OLP) regression was used to test for sonographer and observer biases. RESULTS: The new DICOM-based software was much faster than older bitmap-based software (average measurement time for one scan 3.4 +/- 0.6 minutes versus 8.4 +/- 1.8 minutes, p < 0.0001) but CIMT measurements were larger than those made using the alternative software (+0.02 mm, 95%CI 0.01-0.03 mm). The sixty-frame measurement protocol had worse reproducibility than the six-frame protocol (inter-observer CV 5.1% vs 3.5%, p = 0.004) and inter and intra-observer biases were more pronounced in the sixty-frame than the six-frame results. CONCLUSION: While the use of DICOM-based software significantly reduced analysis time, a ten-fold increase in the number of measurements used to calculate CIMT did not improve reproducibility. In addition, we found that observer biases caused differences in mean CIMT of a magnitude commonly reported as significant in intervention trials. Our results highlight the importance of good study design with concurrent controls and the need to ensure that no observer drift occurs between baseline and follow-up measurements when CIMT is used to monitor the effect of an intervention.