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We report K α x-ray production with a high energy (110 mJ per pulse at 800 nm before compression/15 mJ at 400 nm after compression), high repetition rate (100 Hz), and high pulse contrast (better than 10−9 at 400 nm) laser system. To develop laser-based x-ray sources for biomedical imaging requires to use high-energy and high-power ultrafast laser system where compression is achieved under vacuum. Using this type of laser system, we demonstrate long-term stability of the x-ray yield, conversion efficiency higher than 1.5×10−5 with a Mo target, and the x-ray spot size close to the optical focal spot. This high-repetition K α x-ray source can be very useful for x-ray phase-contrast imaging. [ABSTRACT FROM AUTHOR]

Bacteria use the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) to control biofilm formation and other key phenotypes in response to environmental signals. Changes in oxygen levels can alter c-di-GMP signaling through a family of proteins termed globin coupled sensors (GCS) that contain diguanylate cyclase domains. Previous studies have found that GCS diguanylate cyclase activity is controlled by ligand binding to the heme within the globin domain, with oxygen binding resulting in the greatest increase in catalytic activity. Herein, we present evidence that heme-edge residues control O 2 -dependent signaling in PccGCS, a GCS protein from Pectobacterium carotovorum, by modulating heme distortion. Using enzyme kinetics, resonance Raman spectroscopy, small angle X-ray scattering, and multi-wavelength analytical ultracentrifugation, we have developed an integrated model of the full-length PccGCS tetramer and have identified conformational changes associated with ligand binding, heme conformation, and cyclase activity. Taken together, these studies provide new insights into the mechanism by which O 2 binding modulates activity of diguanylate cyclase-containing GCS proteins., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Emily Weinert reports financial support was provided by National Science Foundation. Emily Weinert reports financial support was provided by Herman Frasch Foundation for Chemical Research. Yilin Liu reports financial support was provided by National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 CYP17A1 (CYP17A1) bound with either of the lyase substrates, 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) are shown to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography-mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions, nor the conditions produced by the cryoradiolysis process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

The human sterol 14α-demethylases (CYP51, CYP is an abbreviation for cytochrome P450) catalyze three-step oxidative removal of 14α-methyl group of lanosterol by first forming an alcohol, then an aldehyde, and finally conducting a CC bond cleavage reaction. This present study utilizes a combination of Resonance Raman spectroscopy and Nanodisc technology to probe the active site structure of CYP51 in the presence of its hydroxylase and lyase substrates. Ligand-binding induced partial low-to-high-spin conversion is observed by applying electronic absorption spectroscopy and Resonance Raman (RR) spectroscopy. This low degree of spin conversion of CYP51 is contributed by the retention of the water ligand coordinated to the heme iron as well as direct interaction between the hydroxyl group of lyase substrate and the iron center. No significant changes in active site structure are found between detergent-stabilized CYP51 and nanodisc-incorporated CYP51, nevertheless, it is demonstrated that nanodisc-incorporated assemblies provide much more well-defined active site RR spectroscopic responses, which induces a larger conversion from low-to-high-spin state in presence of the substrates. Moreover, a positive polar environment around the exogenous diatomic ligand is detected, providing insight into the mechanism of this essential CC bond cleavage reaction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The Cytochrome P450 (CYP450) superfamily has been the subject of intense research for over six decades. Here the HU227 strain of E. coli, lacking the δ-aminolevulinic acid (δ-ALA) synthase gene, was employed, along with [5- 13 C] δ-ALA, in the heterologous expression of P450cam harboring a prosthetic group labeled with 13 C at the four methine carbons (C m ) and pyrrole C α positions. The product was utilized as a proof of principle strategy for defining and refining solution phase active site structure in cytochrome P450cam, providing proton-to-proton distances from 13 C m H to protons on bound substrate or nearby amino acid residues, using short mixing time 2D or 3D NOESY-HMQC methods. The results reveal the interesting finding that 2D 13 C-filtered NOESY-HMQC can be used to obtain distances between protons on labeled 13 C to positions of protons nearby in the active site, confirming the utility of this NMR-based approach to probing active site structure under physiological conditions. Such 13 C-heme-filtered NOE data complement X-ray crystallographic and T1-based NMR measurements; and, may also be of potentially significant utility in furnishing experimental distance constraints in validations of docking routines commonly employed for determining the relative affinities and binding orientations of drug candidates with CYP450s., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interest or personal relationships that influenced or could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The multifunctional cytochrome P450 17A1 (CYP17A1) plays a crucial role in human steroid hormone synthesis (UniProtKB─P05093). It first carries out standard monooxygenase chemistry, converting pregnenolone (PREG) and progesterone (PROG) into 17OH-PREG and 17OH-PROG, utilizing a "Compound I" to initiate hydrogen abstraction and radical recombination in the classic "oxygen rebound" mechanism. Additionally, these hydroxylated products also serve as substrates in a second oxidative cycle which cleaves the 17-20 carbon-carbon bond to form dehydroepiandrosterone and androstenedione, which are key precursors in the generation of powerful androgens and estrogens. Interestingly, in humans, with 17OH-PREG, this so-called lyase reaction is more efficient than with 17OH-PROG, based on K cat / K m values. In the present work, the asparagine residue at 202 position was replaced by serine, an alteration which can affect substrate orientation and control substrate preference for the lyase reaction. First, we report studies of solvent isotope effects for the N202S CYP17A1 mutant in the presence of 17OH-PREG and 17OH-PROG, which suggest that the ferric peroxo species is the predominant catalytically active intermediate in the lyase step. This conclusion is further supported by employing a combination of cryoradiolysis and resonance Raman techniques to successfully trap and structurally characterize the key reaction intermediates, including the peroxo, the hydroperoxo, and the crucial peroxo-hemiketal intermediate. Collectively, these studies show that the mutation causes active site structural changes that alter the H-bonding interactions with the key Fe-O-O fragment and the degree of protonation of the reactive ferric peroxo intermediate, thereby impacting lyase efficiency.

Bifunctional enzymes, which contain two domains with opposing enzymatic activities, are widely distributed in bacteria, but the regulatory mechanism(s) that prevent futile cycling are still poorly understood. The recently described bifunctional enzyme, DcpG, exhibits unusual heme properties and is surprisingly able to differentially regulate its two cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic domains in response to heme gaseous ligands. Mutagenesis of heme-edge residues was used to probe the heme pocket and resulted in decreased O 2 dissociation kinetics, identifying roles for these residues in modulating DcpG gas sensing. In addition, the resonance Raman spectra of the DcpG wild type and heme-edge mutants revealed that the mutations alter the heme electrostatic environment, vinyl group conformations, and spin state population. Using small-angle X-ray scattering and negative stain electron microscopy, the heme-edge mutations were demonstrated to cause changes to the protein conformation, which resulted in altered signaling transduction and enzyme kinetics. These findings provide insights into molecular interactions that regulate DcpG gas sensing as well as mechanisms that have evolved to control multidomain bacterial signaling proteins.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C 17 -C 20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17-C20 bond scission, which is strongly dependent on the presence of cytochrome b 5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b 5 on C-C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b 5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b 5 (Mn-Cyt b 5 ) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe-O mode is observed in the presence of Mn- b 5 , indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe-O-O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cyt b 5 . These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.

Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C 17 -C 20 bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17-OH PREG, not only reveals an initially trapped peroxo-iron intermediate experiencing an H-bond interaction of the 17-OH group with the proximal oxygen of the Fe-O p -O t fragment, facilitating peroxo- attack on the C 20 carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction., (© 2020 Wiley-VCH GmbH.)

Cytochromes P450 bind and cleave dioxygen to generate a potent intermediate compound I, capable of hydroxylating inert hydrocarbon substrates. Cytochrome P450 119, a bacterial cytochrome P450 that serves as a good model system for the study of the intermediate states in the P450 catalytic cycle. CYP119 is found in high temperature and sulfur rich environments. Though the natural substrate and redox partner are still unknown, a potential application of such thermophilic P450s is utilizing them as biocatalysts in biotechnological industry; e.g., the synthesis of organic compounds otherwise requiring hostile environments like extremes of pH or temperature. In the present work the oxygenated complex of this enzyme bound to lauric acid, a surrogate substrate known to have a good binding affinity, was studied by a combination of cryoradiolysis and resonance Raman spectroscopy, to trap and characterize active site structures of the key fleeting enzymatic intermediates, including the peroxo and hydroperoxo species., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A novel family of bacterial hemoproteins named NosP has been discovered recently; its members are proposed to function as nitric oxide (NO) responsive proteins involved in bacterial group behaviors such as quorum sensing and biofilm growth and dispersal. Currently, little is known about molecular activation mechanisms in NosP. Here, functional studies were performed utilizing the distinct spectroscopic characteristics associated with the NosP heme cofactor. NosPs from Pseudomonas aeruginosa ( Pa), Vibrio cholerae ( Vc), and Legionella pneumophila ( Lpg) were studied in their ferrous unligated forms as well as their ferrous CO, ferrous NO, and ferric CN adducts. The resonance Raman (rR) data collected on the ferric forms strongly support the existence of a distorted heme cofactor, which is a common feature in NO sensors. The ferrous spectra exhibit a 213 cm -1 feature, which is assigned to the Fe-N his stretching mode. The Fe-C and C-O frequencies in the spectra of ferrous CO NosP complexes are inversely correlated with relatively similar frequencies, consistent with a proximal histidine ligand and a relatively hydrophobic environment. The rR spectra obtained for isotopically labeled ferrous NO adducts provide evidence of formation of a 5-coordinate NO complex, resulting from proximal Fe-N his cleavage, which is believed to play a role in biological heme-NO signal transduction. Additionally, we found that of the three NosPs studied, Lpg NosP contains the most electropositive ligand binding pocket, while Pa NosP has the most electronegative ligand binding pocket. This pattern is also observed in the measured heme reduction potentials for these three proteins, which may indicate distinct functions for each.

The multifunctional enzyme, cytochrome P450 (CYP17A1), plays a crucial role in the production of androgens, catalyzing two key reactions on pregnenolone (PREG) and progesterone (PROG), the first being a 17-hydroxylation to generate 17-OH PREG and 17-OH PROG, with roughly equal efficiencies. The second is a C-C bond scission or "lyase" reaction in which the C17-C20 bond is cleaved, leading to the eventual production of powerful androgens, whose involvement in the proliferation of prostate cancer has generated intense interest in developing inhibitors of CYP17A1. For humans, the significance of the C-C bond cleavage of 17-OH PROG is lessened, because it is about 50 times less efficient than for 17-OH PREG in terms of k cat /K m . Recognizing the need to clarify relevant reaction mechanisms involved with such transformations, we first report studies of solvent isotope effects, results of which are consistent with a Compound I mediated PROG hydroxylase activity, yet exclude this intermediate as a participant in the formation of androstenedione (AD) via the lyase reaction. This finding is also supported by a combination of cryoreduction and resonance Raman spectroscopy that traps and structurally characterizes the key hemiketal reaction intermediates. Adding to a previous study of PREG and 17-OH PREG metabolism, the current work provides definitive evidence for a more facile protonation of the initially formed ferric peroxo-intermediate for 17-OH PROG-bound CYP17A1, compared to the complex with 17-OH PREG. Importantly, Raman characterization also reveals an H-bonding interaction with the terminal oxygen of the peroxo fragment, rather than with the proximal oxygen, as is present for 17-OH PREG. These factors would favor a diminished lyase activity of the sample with 17-OH PROG relative to the complex with 17-OH PREG, thereby providing a convincing structural explanation for the dramatic differences in activity for these lyase substrates in humans.

CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations on multiple substrates. In its hydroxylase activity, this enzyme adds a hydroxyl group at the 17α position of both pregnenolone and progesterone at approximately equal rates. However, the subsequent 17,20 carbon-carbon scission reaction displays variable substrate specificity in the numerous CYP17A1 isozymes operating in vertebrates, manifesting as different K d and k cat values when presented with 17α-hydroxypregnenlone (OHPREG) versus 17α-hydroxyprogesterone (OHPROG). Here we show that the identity of the residue at position 202 in human CYP17A1, thought to form a hydrogen bond with the A-ring alcohol substituent on the pregnene- nucleus, is a key driver of this enzyme's native preference for OHPREG. Replacement of asparagine 202 with serine completely reverses the preference of CYP17A1, more than doubling the rate of turnover of the OHPROG to androstenedione reaction and substantially decreasing the rate of formation of dehydroepiandrosterone from OHPREG. In a series of resonance Raman experiments, it was observed that, in contrast with the case for the wild-type protein, in the mutant the 17α alcohol of OHPROG tends to form a H-bond with the proximal rather than terminal oxygen of the oxy-ferrous complex. When OHPREG was a substrate, the mutant enzyme was found to have a H-bonding interaction with the proximal oxygen that is substantially weaker than that of the wild type. These results demonstrate that a single-point mutation in the active site pocket of CYP17A1, even when far from the heme, has profound effects on steroidogenic selectivity in androgen biosynthesis.

The rate-limiting step in the steroid synthesis pathway is catalyzed by CYP11A1 through three sequential reactions. The first two steps involve hydroxylations at positions 22 and 20, generating 20(R),22(R)-dihydroxycholesterol (20R,22R-DiOHCH), with the third stage leading to a C20-C22 bond cleavage, forming pregnenolone. This work provides detailed information about the active site structure of CYP11A1 in the resting state and substrate-bound ferric forms as well as the CO-ligated adducts. In addition, high-quality resonance Raman spectra are reported for the dioxygen complexes, providing new insight into the status of Fe-O-O fragments encountered during the enzymatic cycle. Results show that the three natural substrates of CYP11A1 have quite different effects on the active site structure, including variations of spin state populations, reorientations of heme peripheral groups, and, most importantly, substrate-mediated distortions of Fe-CO and Fe-O 2 fragments, as revealed by telltale shifts of the observed vibrational modes. Specifically, the vibrational mode patterns observed for the Fe-O-O fragments with the first and third substrates are consistent with H-bonding interactions with the terminal oxygen, a structural feature that tends to promote O-O bond cleavage to form the Compound I intermediate. Furthermore, such spectral data are acquired for complexes with the natural redox partner, adrenodoxin (Adx), revealing protein-protein-induced active site structural perturbations. While this work shows that Adx has an only weak effect on ferric and ferrous CO states, it has a relatively stronger impact on the Fe-O-O fragments of the functionally relevant oxy complexes.

DGCR8 is the RNA-binding partner of the nuclease Drosha. Their complex (the "Microprocessor") is essential for processing of long, primary microRNAs (pri-miRNAs) in the nucleus. Binding of heme to DGCR8 is essential for pri-miRNA processing. On the basis of the split Soret ultraviolet-visible (UV-vis) spectrum of ferric DGCR8, bis-thiolate sulfur (cysteinate, Cys(-)) heme iron coordination of DGCR8 heme iron was proposed. We have characterized DGCR8 heme ligation using the Δ276 DGCR8 variant and combined electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), electron nuclear double resonance, resonance Raman, and electronic absorption spectroscopy. These studies indicate DGCR8 bis-Cys heme iron ligation, with conversion from bis-thiolate (Cys(-)/Cys(-)) axial coordination in ferric DGCR8 to bis-thiol (CysH/CysH) coordination in ferrous DGCR8. Pri-miRNA binding does not perturb ferric DGCR8's optical spectrum, consistent with the axial ligand environment being separated from the substrate-binding site. UV-vis absorption spectra of the Fe(II) and Fe(II)-CO forms indicate discrete species exhibiting peaks with absorption coefficients substantially larger than those for ferric DGCR8 and that previously reported for a ferrous form of DGCR8. Electron-nuclear double resonance spectroscopy data exclude histidine or water as axial ligands for ferric DGCR8 and favor bis-thiolate coordination in this form. UV-vis MCD and near-infrared MCD provide data consistent with this conclusion. UV-vis MCD data for ferrous DGCR8 reveal features consistent with bis-thiol heme iron coordination, and resonance Raman data for the ferrous-CO form are consistent with a thiol ligand trans to the CO. These studies support retention of DGCR8 cysteine coordination upon reduction, a conclusion distinct from those of previous studies of a different ferrous DGCR8 isoform.

Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second CC lyase step, at the expense of glucocorticoid production. Cytochrome b5 (cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced ∼5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal FeS vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b5 has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis., (Published by Elsevier Inc.)

Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C17-C20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon-carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences.

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.

The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or β hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe-histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or β chains, prior to the quaternary R-T and T-R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe-His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem. 2002, 98, 149). Experimentally, the νFeHis evolution is faster for β than for α chains, and pump-probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 μs for β but not for α hemes, an interval previously shown to be the first step in the R-T transition. In the α chain νFeHis dropped sharply at 20 μs, the final step in the R-T transition. The time courses are fully consistent with recent computational mapping of the R-T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.

CYP19A1, or aromatase, a cytochrome P450 responsible for estrogen biosynthesis in humans, is an important therapeutic target for the treatment of breast cancer. There is still controversy surrounding the identity of reaction intermediate that catalyzes carbon-carbon scission in this key enzyme. Probing the oxy-complexes of CYP19A1 poised for hydroxylase and lyase chemistries using resonance Raman spectroscopy and drawing a comparison with CYP17A1, we have found no significant difference in the frequencies or isotopic shifts for these two steps in CYP19A1. Our experiments implicate the involvement of Compound I in the terminal lyase step of CYP19A1 catalysis.

An important function of steroidogenic cytochromes P450 is the transformation of cholesterol to produce androgens, estrogens, and the corticosteroids. The activities of cytochrome P450c17 (CYP17) are essential in sex hormone biosynthesis, with severe developmental defects being a consequence of deficiency or mutations. The first reaction catalyzed by this multifunctional P450 is the 17α-hydroxylation of pregnenolone (PREG) to 17α-hydroxypregnenolone (17-OH PREG) and progesterone (PROG) to 17α-hydroxyprogesterone (17-OH PROG). The hydroxylated products then either are used for production of corticoids or undergo a second CYP17 catalyzed transformation, representing the first committed step of androgen formation. While the hydroxylation reactions are catalyzed by the well-known Compound I intermediate, the lyase reaction is believed to involve nucleophilic attack of the earlier peroxo- intermediate on the C20-carbonyl. Herein, resonance Raman (rR) spectroscopy reveals that substrate structure does not impact heme structure for this set of physiologically important substrates. On the other hand, rR spectra obtained here for the ferrous CO adducts with these four substrates show that substrates do interact differently with the Fe-C-O fragment, with large differences between the spectra obtained for the samples containing 17-OH PROG and 17-OH PREG, the latter providing evidence for the presence of two Fe-C-O conformers. Collectively, these results demonstrate that individual substrates can differentially impact the disposition of a heme-bound ligand, including dioxygen, altering the reactivity patterns in such a way as to promote preferred chemical conversions, thereby avoiding the profound functional consequences of unwanted side reactions.

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 (LS) to high-spin (HS), 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 ν 3 "spin state marker" bands. Here, employing a reference cytochrome P450 (CYP101), the intensities of the ν 3 modes (I LS ) and (I HS ) 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 ν 3 modes. 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 Cytochromes 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.

Purpose: Methods of reducing respiratory motion blurring in cone-beam CT (CBCT) have been limited to lung where soft tissue contrast is large. Respiration-correlated cone-beam CT uses slow continuous gantry rotation but image quality is limited by uneven projection spacing. This study investigates the efficacy of a novel gated CBCT technique., Methods: In gated CBCT, the linac is programmed such that gantry rotation and kV image acquisition occur within a gate around end expiration and are triggered by an external respiratory monitor. Standard CBCT and gated CBCT scans are performed in 22 patients (11 thoracic, 11 abdominal) and a respiration-correlated CT (RCCT) scan, acquired on a standard CT scanner, from the same day serves as a criterion standard. Image quality is compared by calculating contrast-to-noise ratios (CNR) for tumors in lung, gastroesophageal junction (GEJ) tissue, and pancreas tissue, relative to surrounding background tissue. Congruence between the object in the CBCT images and that in the RCCT is measured by calculating the optimized normalized cross-correlation (NCC) following CBCT-to-RCCT rigid registrations., Results: Gated CBCT results in reduced motion artifacts relative to standard CBCT, with better visualization of tumors in lung, and of abdominal organs including GEJ, pancreas, and organs at risk. CNR of lung tumors is larger in gated CBCT in 6 of 11 cases relative to standard CBCT. A paired two-tailed t-test of lung patient mean CNR shows no statistical significance (p = 0.133). In 4 of 5 cases where CNR is not increased, lung tumor motion observed in RCCT is small (range 1.3-5.2 mm). CNR is increased and becomes statistically significant for 6 out of 7 lung patients with > 5 mm tumor motion (p = 0.044). CNR is larger in gated CBCT in 5 of 7 GEJ cases and 3 of 4 pancreas cases (p = 0.082 and 0.192). Gated CBCT yields improvement with lower NCC relative to standard CBCT in 10 of 11, 7 of 7, and 3 of 4 patients for lung, GEJ, and pancreas images, respectively (p = 0.0014, 0.0030, 0.165)., Conclusions: Gated CBCT reduces image blurring caused by respiratory motion. The gated gantry rotation yields uniformly and closely spaced projections resulting in improved reconstructed image quality. The technique is shown to be applicable to abdominal sites, where image contrast of soft tissues is low.

Resonance Raman (RR) spectroscopy is used to help define active site structural responses of nanodisc-incorporated CYP3A4 to the binding of three substrates: bromocriptine (BC), erythromycin (ERY), and testosterone (TST). We demonstrate that nanodisc-incorporated assemblies reveal much more well-defined active site RR spectroscopic responses as compared to those normally obtained with the conventional, detergent-stabilized, sampling strategies. While ERY and BC are known to bind to CYP3A4 with a 1:1 stoichiometry, only the BC induces a substantial conversion from low- to high-spin state, as clearly manifested in the RR spectra acquired herein. The third substrate, TST, displays significant homotropic interactions within CYP3A4, the active site binding up to 3 molecules of this substrate, with the functional properties varying in response to binding of individual substrate molecules. While such behavior seemingly suggests the possibility that each substrate binding event induces functionally important heme structural changes, up to this time spectroscopic evidence for such structural changes has not been available. The current RR spectroscopic studies show clearly that accommodation of different size substrates, and different loading of TST, do not significantly affect the structure of the substrate-bound ferric heme. However, it is here demonstrated that the nature and number of bound substrates do have an extraordinary influence on the conformation of bound exogenous ligands, such as CO or dioxygen and its reduced forms, implying an effective mechanism whereby substrate structure can impact reactivity of intermediates so as to influence function, as reflected in the diverse reactivity of this drug metabolizing cytochrome.

In view of the potent oxidizing strength of cytochrome P450 intermediates, it is not surprising that certain substrates can give rise to reactive species capable of attacking the heme or critical distal-pocket protein residues to irreversibly modify the enzyme in a process known as mechanism-based (MB) inactivation, a result that can have serious physiological consequences leading to adverse drug-drug interactions and toxicity. While methods exist to document the attachment of these substrate fragments, it is more difficult to gain insight into the structural basis for the altered functional properties of these modified enzymes. In response to this pressing need to better understand MB inhibition, we here report the first application of resonance Raman spectroscopy to study the inactivation of a truncated form of mammalian CYP2B4 by the acetylenic inhibitor 4-(tert-butyl)phenylacetylene, whose activated form is known to attach to the distal-pocket T302 residue of CYP2B4.

Time-resolved resonance Raman (RR) spectra are reported for hemoglobin (Hb) tetramers, in which the alpha and beta chains are selectively substituted with mesoheme. The Soret absorption band shift in mesoheme relative to protoheme permits chain-selective recording of heme RR spectra. The evolution of these spectra following HbCO photolysis shows that the geminate recombination rates and the yields are the same for the two chains, consistent with recent results on (15)N-heme isotopomer hybrids. The spectra also reveal systematic shifts in the deoxyheme nu (4) and nu (Fe-His) RR bands, which are anticorrelated. These shifts are resolved for the successive intermediates in the protein structure, which have previously been determined from time-resolved UV RR spectra. Both chains show Fe-His bond compression in the immediate photoproduct, which relaxes during the formation of the first intermediate, R(deoxy) (0.07 micros), in which the proximal F-helix is proposed to move away from the heme. Subsequently, the Fe-His bond weakens, more so for the alpha chains than for the beta chains. The weakening is gradual for the beta chains, but is abrupt for the alpha chains, coinciding with completion of the R-T quaternary transition, at 20 micros. Since the transition from fast- to slow-rebinding Hb also occurs at 20 micros, the drop in the alpha chain nu (Fe-His) supports the localization of ligation restraint to tension in the Fe-His bond, at least in the alpha chains. The mechanism is more complex in the beta chains.

Hemoglobin (Hb) is an allosteric tetrameric protein made up of alphabeta heterodimers. The alpha and beta chains are similar, but are chemically and structurally distinct. To investigate dynamical differences between the chains, we have prepared tetramers in which the chains are isotopically distinguishable, via reconstitution with (15)N-heme. Ligand recombination and heme structural evolution, following HbCO dissociation, was monitored with chain selectivity by resonance Raman (RR) spectroscopy. For alpha but not for beta chains, the frequency of the nu(4) porphyrin breathing mode increased on the microsecond time scale. This increase is a manifestation of proximal tension in the Hb T-state, and its time course is parallel to the formation of T contacts, as determined previously by UVRR spectroscopy. Despite the localization of proximal constraint in the alpha chains, geminate recombination was found to be equally probable in the two chains, with yields of 39 +/- 2%. We discuss the possibility that this equivalence is coincidental, in the sense that it arises from the evolutionary pressure for cooperativity, or that it reflects mechanical coupling across the alphabeta interface, evidence for which has emerged from UVRR studies of site mutants.

Resonance Raman (RR) studies of intermediates generated by cryoreduction of the oxyferrous complex of the D251N mutant of cytochrome P450(cam) (CYP101) are reported. Owing to the fact that proton delivery to the active site is hindered in this mutant, the unprotonated peroxo-ferric intermediate is observed as the primary species after radiolytic reduction of the oxy-complex in frozen solutions at 77 K. In as much as previous EPR and ENDOR studies have shown that annealing of this species to approximately 180 K results in protonation of the distal oxygen atom to form the hydroperoxo intermediate, this system has been exploited to permit direct RR interrogation of the changes in the Fe-O and O-O bonds caused by the reduction and subsequent protonation. Our results show that the nu(O-O) mode decreases from a superoxo-like frequency near approximately 1130 cm(-1) to 792 cm(-1) upon reduction. The latter frequency, as well as its lack of sensitivity to H/D exchange, is consistent with heme-bound peroxide formulation. This species also exhibits a nu(Fe-O) mode, the 553 cm(-1) frequency of which is higher than that observed for the nonreduced oxy P450 precursor (537 cm(-1)), implying a strengthened Fe-O linkage upon reduction. Upon subsequent protonation, the resulting Fe-O-OH fragment exhibits a lowered nu(O-O) mode at 774 cm(-1), whereas the nu(Fe-O) increases to 564 cm(-1). Both modes exhibit a downshift upon H/D exchange, as expected for a hydroperoxo-ferric formulation. These experimental RR data are compared with those previously acquired for the wild-type protein, and the shifts observed upon reduction and subsequent protonation are discussed with reference to theoretical predictions.

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 nu(Fe-N(His)), nu(Fe-C), and nu(C-O) modes. In fact, the apparent lack of an effect on these key vibrational parameters of the Fe-N(His), 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.

Resonance Raman spectra are reported for substrate-free and camphor-bound cytochrome P450cam and its isotopically labeled analogues that have been reconstituted with protoheme derivatives that bear -CD(3) groups at the 1, 3, 5, and 8-positions (d12-protoheme) or deuterated methine carbons (d4-protoheme). In agreement with previous studies of this and similar enzymes, substrate binding induces changes in the high frequency and low frequency spectral regions, with the most dramatic effect in the low frequency region being activation of a new mode near 367 cm(-1). This substrate-activated mode had been previously assigned as a second "propionate bending" mode (Chen et al., Biochemistry, 2004, 43, 1798-1808), arising in addition to the single propionate bending mode observed for the substrate-free form at 380 cm(-1). In this work, this newly activated mode is observed to shift by 8 cm(-1) to lower frequency in the d12-protoheme reconstituted enzyme (i.e., the same shift as that observed for the higher frequency "propionate bending" mode) and is therefore consistent with the suggested assignment. However, the newly acquired data for the d4-protoheme substituted analogue also support an earlier alternate suggestion (Deng et al., Biochemistry, 1999, 38, 13699-13706) that substrate binding activates several heme out-of-plane modes, one of which (gamma(6)) is accidentally degenerate with the 367 cm(-1) propionate bending mode. Finally, the study of the enzyme reconstituted with the protoheme-d4, which shifts the macrocycle nu(10) mode, has now allowed a definitive identification of the vinyl C=C stretching modes.

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 nu(O-O) stretching mode in the native systems. The present work was undertaken to explore the utility of cobalt substitution to enhance both the nu(Co-O) and nu(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 (16)O(2) and (18)O(2) to reveal the nu(M-O) and nu(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 studies of P450 2B4 are reported for the substrate-free form and when bound to the substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT), the latter representing a substrate capable of inducing an especially effective conversion to the high-spin state. In addition to studies of the ferric resting state, spectra are acquired for the ferrous CO ligated form. Importantly, for the first time, the RR technique is effectively applied to interrogate the changes in active site structure induced by binding of cytochrome P450 reductase (CPR) and Mn(III) cytochrome b 5 (Mn cyt b 5); the manganese derivative of cyt b 5 was employed to avoid spectroscopic interferences. The results, consistent with early work on mammalian P450s, demonstrate that substrate structure has minimal effects on heme structure or the FeCO fragment of the ferrous CO derivatives. Similarly, the data indicate that the protein is flexible and that substrate binding does not exert significant strain on the heme peripheral groups, in contrast to P450 cam, where substantial effects on heme peripheral groups are seen. However, significant differences are observed in the RR spectra of P450 2B4 when bound with the different redox partners, indicating that the heme structure is clearly sensitive to perturbations near the proximal heme binding site. The most substantial changes are displacements of the peripheral vinyl groups toward planarity with the heme macrocycle by cyt b 5 but away from planarity by CPR. These changes can have an impact on heme reduction potential. Most interestingly, these RR results support an earlier observation that the combination of benzphetamine and cyt b 5 binding produce a synergy leading to unique active site structural changes when both are bound.

Heme proteins that have been reconstituted with certain hemins may contain substantial fractions of a minor component in which the orientation of the heme in the folded pocket differs from the major ("native") conformation by a 180 degrees rotation about the alpha-gamma meso axis. In fact, this minor component has also been shown to exist in some native proteins, including several mammalian globins. While resonance Raman spectroscopy has emerged as a powerful probe of active site structure of heme proteins, no systematic study has yet been undertaken to elucidate the specific spectral changes associated with this disorder. In the present work, combined analyses of the temporal behavior of both NMR and RR data sets have been completed to permit the extraction of a unique RR spectrum for the disoriented form, documenting rather dramatic changes associated with this rotational disorder. In addition, the use of protohemes bearing selectively deuterated peripheral methyl groups has permitted the association of the observed modes with specific fragments of the heme residing in the reversed orientation. The studies conducted here clearly illustrate the exquisite sensitivity of low frequency heme deformation modes to altered protein-heme interactions., (2007 Wiley Periodicals, Inc)

In an attempt to gain further insight into the nature of the low frequency vibrational modes of hemoglobin and its isolated subunits, a comprehensive study of several different isotopically labeled analogues has been undertaken and is reported herein. Specifically, the resonance Raman spectra, between 200 and 500 cm(-1), are reported for the deoxy and ligated (CO and O2) forms of the isolated alpha and beta subunits containing the natural abundance or various deuterated analogues of protoheme. The deuterated protoheme analogues studied include the 1,3,5,8-C2H3-protoheme (d12- protoheme), the 1,3-C2H3-protoheme (1,3-d6-protoheme), the 5,8-C2H3-protoheme (5,8-d6-protoheme), and the meso-C2H4-protoheme (d4-protoheme). The entire set of acquired spectra has been analyzed using a deconvolution procedure to help correlate the shifted modes with their counterparts in the spectra of the native forms. Interestingly, modes previously associated with so-called vinyl bending modes or propionate deformation modes are shown to be quite sensitive to deuteration of the peripheral methyl groups of the macrocycle, shifting by up to 12-15 cm(-1), revealing their complex nature. Of special interest is the fact that shifts observed for the 1,3-d6- and 5,8-d6-protoheme analogues confirm the fact that certain modes are associated with a given portion of the macrocycle; i.e., only certain modes shift upon deuteration of the 1 and 3 methyl groups, while others shift upon deuteration of the 5 and 8 methyl groups. Compared with the spectra previously reported for the corresponding myoglobin derivatives, the data reported here reveal the appearance of several additional features that imply splitting of modes associated with the propionate groups or that are indicative of greater distortion of the heme prosthetic groups., ((c) 2006 Wiley Periodicals, Inc.)

Resonance Raman spectra are reported for a series of systematically deuterated analogues of myoglobin in its deoxy state as well as for its CO and O(2) adducts. Specifically, the myoglobin samples studied are those that have been reconstituted with deuterated protoheme analogues. These include the methine deuterated, protoheme-d4; analogue bearing C(2)H(3) groups at the 1, 3, 5, and 8 positions, protoheme-d12; the species bearing C(2)H(3) groups at the 1 and 3 positions only, 1,3-protoheme-d6; and the species bearing C(2)H(3) groups at the 5 and 8 positions only, 5,8-protoheme-d6. While the results are generally consistent with previously reported data for synthetic metalloporphyrin models and previous studies of labeled heme proteins, the high-quality low-frequency RR data reported here reveal several important aspects of these low-frequency modes. Of special interest is the fact that, using the two d6-protoheme analogues, it is shown that certain modes are apparently localized on particular pyrrole rings, while others are localized on different rings; i.e., several of these low-frequency modes are localized on one side of the heme.