Resonance Raman Spectroscopic Studies of Hydroperoxo Derivatives of Cobalt-substituted Myoglobin

Recent progress in generating and stabilizing reactive heme protein enzymatic intermediates by cryoradiolytic reduction has prompted application of a range of spectroscopic approaches to effectively interrogate these species. The impressive potential of resonance Raman spectroscopy for characterizing such samples has been recently demonstrated in a number of studies of peroxo- and hydroperoxo-intermediates. While it is anticipated that this approach can be productively applied to the wide range of heme proteins whose reaction cycles naturally involve these peroxo- and hydroperoxo-intermediates, one limitation that sometimes arises is the lack of enhancement of the key intraligand ν(O–O) stretching mode in the native systems. The present work was undertaken to explore the utility of cobalt substitution to enhance both the ν(Co–O) and ν(O–O) modes of the CoOOH fragments of hydroperoxo forms of heme proteins bearing a trans–axial histidine linkage. Thus, having recently completed RR studies of hydroperoxo myoglobin, attention is now turned to its cobalt-substituted analogue. Spectra are acquired for samples prepared with 16O2 and 18O2 to reveal the ν(M–O) and ν(O–O) modes, the latter indeed being observed only for the cobalt-substituted proteins. In addition, spectra of samples prepared in deuterated solvents were also acquired, providing definitive evidence for the presence of the hydroperoxo-species

Resonance Raman Interrogation of the Consequences of Heme Rotational Disorder in Myoglobin and its Ligated Derivatives

Resonance Raman spectroscopy is employed to characterize heme site structural changes arising from conformational heterogeneity in deoxyMb and ligated derivatives, i.e., the ferrous CO (MbCO) and ferric cyanide (MbCN) complexes. The spectra for the reversed forms of these derivatives have been extracted from the spectra of reconstituted samples. Dramatic changes in the low-frequency spectra are observed, where newly observed RR modes of the reversed forms are assigned using protohemes that are selectively deuterated at the four methyl groups or at the four methine carbons. Interestingly, while substantial changes in the disposition of the peripheral vinyl and propionate groups can be inferred from the dramatic spectral shifts, the bonds to the internal histidyl imidazole ligand and those of the Fe−CO and Fe−CN fragments are not significantly affected by the heme rotation, as judged by lack of significant shifts in the ν(Fe−NHis), ν(Fe−C), and ν(C−O) modes. In fact, the apparent lack of an effect on these key vibrational parameters of the Fe−NHis, Fe−CO, and Fe−CN fragments is entirely consistent with previously reported equilibrium and kinetic studies that document virtually identical functional properties for the native and reversed forms

Using Resonance Raman Cross-section Data to Estimate the Spin State Populations of Cytochromes P450

The cytochromes P450 (CYPs) are heme proteins responsible for the oxidation of xenobiotics and pharmaceuticals and the biosynthesis of essential steroid products. In all cases, substrate binding initiates the enzymatic cycle, converting ferric low-spin to high-spin state, with the efficiency of the conversion varying widely for different substrates, so documentation of this conversion for a given substrate is an important objective. Resonance Raman (rR) spectroscopy can effectively yield distinctive frequencies for the ν3 ‘spin state marker’ bands. Here, employing a reference cytochrome P450 (CYP101), the intensities of the ν3 modes (ILS) and (IHS) relative to an internal standard (sodium sulfate) yield relative populations for the two spin states; i.e., a value of 1.24 was determined for the ratio of the relative cross sections for the ν3 modes. The use of this value was then shown to permit a reliable calculation of relative populations of the two spin states from rR spectra of several other CYPs P450. The importance of this work is that, using this information, it is now possible to conveniently document by rR the spin state population without conducting separate experiments requiring different analytical methods, instrumentation, and additional sample

Research on the structural performance of large rocket booster subjected to longitudinal excitations

Dynamic structural behavior of large booster rocket subjected to longitudinal excitations - analysis of theoretical mode

Resonance Raman Spectroscopy Reveals pH-Dependent Active Site Structural Changes of Lactoperoxidase Compound 0 and Its Ferryl Heme O–O Bond Cleavage Products

The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), is binding of hydrogen peroxide to the ferric resting state to form a ferric-hydroperoxo intermediate designated as Compound 0, the residual proton temporarily associating with the distal pocket His109 residue. Upon delivery of this “stored” proton to the hydroperoxo fragment, it rapidly undergoes O–O bond cleavage, thereby thwarting efforts to trap it using rapid mixing methods. Fortunately, as shown herein, both the peroxo and the hydroperoxo (Compound 0) forms of LPO can be trapped by cryoradiolysis, with acquisition of their resonance Raman (rR) spectra now permitting structural characterization of their key Fe–O–O fragments. Studies were conducted under both acidic and alkaline conditions, revealing pH-dependent differences in relative populations of these intermediates. Furthermore, upon annealing, the low pH samples convert to two forms of a ferryl heme O–O bond-cleavage product, whose ν(Fe═O) frequencies reflect substantially different Fe═O bond strengths. In the process of conducting these studies, rR structural characterization of the dioxygen adduct of LPO, commonly called Compound III, has also been completed, demonstrating a substantial difference in the strengths of the Fe–O linkage of the Fe–O–O fragment under acidic and alkaline conditions, an effect most reasonably attributed to a corresponding weakening of the trans-axial histidyl imidazole linkage at lower pH. Collectively, these new results provide important insight into the impact of pH on the disposition of the key Fe–O–O and Fe═O fragments of intermediates that arise in the enzymatic cycles of LPO, other mammalian peroxidases, and related proteins

Strategic Transfer in Logical Abilities in Children Playing Mastermind and an Analogue

Strategy development and the use of strategy as a mechanism of transfer was examined in sixty elementary students while playing the logical deduction game Mastermind and a familiar analogue. In the first couple of two-way ANOVAs subjects showed that they are in fact learning or developing a task-specific strategy that can be applied across the two types of games regardless of which game was in the target position of a transfer paradigm. This suggests that subjects were able to focus on structural similarities rather than surface features and apply what was learned between the game isomorphs. Both the third and fourth ANOVAs indicate that strategic transfer did occur between the Family Dinner Table game and Mastermind game, when mastermind was in the target position of a transfer paradigm. This suggests that strategies can be used as a mechanism in transfer

Strategic Transfer in Logical Abilities in Children Playing Mastermind and an Analogue

Strategy development and the use of strategy as a mechanism of transfer was examined in sixty elementary students while playing the logical deduction game Mastermind and a familiar analogue. In the first couple of two-way ANOVAs subjects showed that they are in fact learning or developing a task-specific strategy that can be applied across the two types of games regardless of which game was in the target position of a transfer paradigm. This suggests that subjects were able to focus on structural similarities rather than surface features and apply what was learned between the game isomorphs. Both the third and fourth ANOVAs indicate that strategic transfer did occur between the Family Dinner Table game and Mastermind game, when mastermind was in the target position of a transfer paradigm. This suggests that strategies can be used as a mechanism in transfer

An Information Base For PM TRADE\u27s Technology Investment Strategy

Report on management strategy for technology investment created for U.S. Army, Project Manager for Training Devices, to be used to communicate and coordinate technology initiatives with industry, academia, and other government organizations