Spectral Characterization of Cytochromes P450 Active Site and Catalytic Intermediates

Cytochromes P450 (P450s) have been the subject of intense research for over six decades. Though it is widely accepted that a highly reactive Fe(IV)=O π-cation radical, or the so called compound I, facilitates the oxidation of relatively inert hydrocarbons, spectroscopic characterization of this putative intermediate has eluded detection under turnover conditions, presumably due to its very short lifetime. In this work, chemically inert substrates of P450s have been utilized in a new approach to capture and stabilize this transient intermediate and characterize it with resonance Raman (RR) spectroscopy, which is a well established tool for studying heme proteins. Specifically, perfluorodecanoic acid has been utilized as an inert surrogate substrate of a thermophilic cytochrome P450 designated CYP119 and RR and cryoradiolysis methods were employed to characterize the enzymatic intermediates under turnover conditions. In a separate project, a recent and more efficient approach for the isotopic labeling of the prosthetic group in heme proteins has been exploited to produce a 13C labeled analogue of the soluble bacterial cytochrome P450cam (P450cam). Briefly, the HU227 strain of E. coli that lacks the δ-aminolevulinic acid (δ-ALA) synthase gene was employed in the heterologous expression of P450cam harboring a prosthetic group labeled with 13C at the Cm and Cα positions by growing cells in the presence of [5-13C] δ-ALA, which was synthesized in four steps from [2-13C] glycine. This system has been utilized as proof of principle for the strategy of defining active site structure in mammalian cytochromes P450 using NMR methods to furnish necessary experimental restrictions in docking routines, which are commonly employed in determining the relative affinities of drug candidates. Noting that few crystal structures of substrate bound complexes of drug metabolizing P450s exist, a truncated CYP2D6 gene has been designed following a recently published procedure and efforts were made to heterologously express a selectively13C enriched analogue of this important drug metabolizing enzyme

Defining Resonance Raman Spectral Responses to Substrate Binding by Cytochrome P450 from \u3cem\u3ePseudomonas putida\u3c/em\u3e

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 -CD3 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 (γ6) 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 ν10 mode, has now allowed a definitive identification of the vinyl CC stretching modes

Similar cellular responses after treatment with either praziquantel or oxamniquine in Schistosoma mansoni infection.

The effect of treatment with either oxamniquine or praziquantel on S.mansoni specific IFN-gamma, IL-4, IL-5 and IL-10 was compared on PBMC which were collected pretreatment, 6 and 18 weeks post treatment. Using sandwich ELISA on the supernatants harvested from the PBMC stimulation by crude S. mansoni SEA and SWAP antigens after 5 days the levels of PBMC proliferation and cytokine production were similar according to treatment with either praziquantel or oxamniquine. Before treatment, infected groups showed low ratios, of IL-4:IFN-gamma, IL-5:IFNgamma and IL-10:IFN-gamma, indicating that IFN-gamma was high in the infected individuals. The general increase in immuno-modulation was observed post-treatment with elevated immune reactivity and cytokine production in both treatment groups. Treatment induced significant increases in levels of IL-4 (p < 0.05), IL-5 (p < 0.0001) and IL-10 (p < 0.05) cytokines 6 and 18 weeks after treatment. There were no significant differences in the increase in IL-4, IL-5 and IL-10 between children treated with praziquantel or oxamniquine. Pre-treatment IFN-gamma and IL-5 levels were positively correlated with infection (p < 0.001), while post treatment IL-4 cytokine levels were negatively correlated with baseline infection status (p < 0.001). The results suggest that treatment-induced immune responses are similar for both common anti-schistosome drugs praziquantel or oxamniquine having similar and immunizing effect

COVID-19 Reflection from Biochemical and Scriptural Perspectives

At the close of the year 2019, reports of a contagious novel virus that causes pneumonia-like symptoms emerged in Wuhan, China. The viral disease, now famously known as coronavirus disease 2019 (COVID-19) has since risen to the level of a global pandemic with over 6.5 million reported cases and almost 400 thousands deaths worldwide. The global scientific community is working around the clock investigating possible control measures, such as development and testing of therapeutic drugs and vaccines. Beside all these advances in technology and biochemical knowledge, is there anything else we can learn from this pandemic? Is it enough that we have some understanding of the viral structure, or the proteins involved in the infection process, or that we have promising vaccine candidates. What else can we learn? What does this mean to a Christian professor of biochemistry, to a chemistry student, to a Taylor Alumni and to all of us who are called by the name of Jesus Christ? In this talk I will share some thoughts on the current pandemic in light of what the scripture says. I invite you to join me in reflecting on this crisis that has essentially shut down our world as we know it

Resonance Raman Spectroscopy of Cytochrome P450cam

Substrate binding in Cytochrome P450 results in the activation of a new mode near 367 cm-1 in the low frequency resonance Raman spectrum. This substrate-activated mode had been previously assigned as a second propionate bending mode, arising in addition to the single propionate bending mode observed for the substrate-free form at 380 cm-1. In the present work, results of resonance Raman studies of the substrate-free and camphor-bound cytochrome P450cam and its isotopically labeled analogues that have been reconstituted with protoheme derivatives that bear -CD3 groups at the 1,3 ,5 and 8-positions (dl2-protoheme) or deuterated methine carbons (d4-protoheme) are discussed. 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 that substrate binding activates several heme out-of-plane modes, one of which ( Y6) is accidentally degenerate with the 367 cm-1 propionate bending mode. In its catalytic cycle, Cytochrome P450 binds and cleaves molecular oxygen to generate a yet to be characterized potent intermediate capable of hydroxylating even relatively inert hydrocarbon substrates. Characterization of this fleeting intermediate by resonance Raman spectroscopy has proven elusive. Utilization of a chemically inert substrate for Cytochrome P450cam (P450cam) might perhaps increase the lifetime of certain fleeting intermediates in the P450cam enzymatic catalytic cycle, possibly facilitating their subsequent characterization by resonance Raman spectroscopy. In this work the binding ofF-adamantanol as an inert substrate for Cytochrome P450cam are summarized

Heme-containing proteins in the Malaria Parasite

Malaria is an infectious disease caused by the parasite, Plasmodium Falciparum. Heme, an iron-containing prosthetic group plays an important and yet poorly understood role in the parasite life cycle. Several genes coding for putative heme-binding proteins have been observed in the parasite genome. Functional characterization of these heme-binding proteins could possibly lead to identification of possible therapeutic targets

Resonance Raman Spectroscopic Studies of Peroxo and Hydroperoxo Intermediates in Lauric Acid (LA)-Bound Cytochrome P450 119

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

Similar cellular responses after treatment with either praziquantel or oxamniquine in Schistosoma mansoni infection.

The effect of treatment with either oxamniquine or praziquantel on S.mansoni specific IFN-gamma, IL-4 , IL-5 and IL-10 was compared on PBMC which were collected pre-treatment, 6 and 18 weeks post treatment. Using sandwich ELISA on the supernatants harvested from the PBMC stimulation by crude S. mansoni SEA and SWAP antigens after 5 days the levels of PBMC proliferation and cytokine production were similar according to treatment with either praziquantel or oxamniquine. Before treatment, infected groups showed low ratios, of IL-4:IFN-gamma, IL-5:IFN-gamma and IL-10:IFN-gamma, indicating that IFN-gamma was high in the infected individuals. The general increase in immuno-modulation was observed post-treatment with elevated immune reactivity and cytokine production in both treatment groups. Treatment induced significant increases in levels of IL-4 (p<0.05), IL-5 (p<0.0001) and IL-10 (p<0.05) cytokines 6 and 18 weeks after treatment. There were no significant differences in the increase in IL-4, IL-5 and IL-10 between children treated with praziquantel or oxamniquine. Pre-treatment IFN-gamma and IL-5 levels were positively correlated with infection (p<0.001), while post treatment IL-4 cytokine levels were negatively correlated with baseline infection status (p<0.001). The Results suggest that treatment-induced immune responses are similar for both commonanti-schistosome drugs praziquantel or oxamniquine having similar and immunizing effect

Spectroscopic and Mutagenesis Studies of Human PGRMC1

Progesterone receptor membrane component 1 (PGRMC1) is a 25 kDa protein with an N-terminal transmembrane domain and a putative C-terminal cytochrome b5 domain. Heme-binding activity of PGRMC1 has been shown in various homologues of PGRMC1. Although the general definition of PGRMC1 is as a progesterone receptor, progesterone-binding activity has not been directly demonstrated in any of the purified PGRMC1 proteins fully loaded with heme. Here, we show that the human homologue of PGRMC1 (hPGRMC1) binds heme in a five-coordinate (5C) high-spin (HS) configuration, with an axial tyrosinate ligand, likely Y95. The negatively charged tyrosinate ligand leads to a relatively low redox potential of approximately −331 mV. The Y95C or Y95F mutation dramatically reduces the ability of the protein to bind heme, supporting the assignment of the axial heme ligand to Y95. On the other hand, the Y95H mutation retains ∼90% of the heme-binding activity. The heme in Y95H is also 5CHS, but it has a hydroxide axial ligand, conceivably stabilized by the engineered-in H95 via an H-bond; CO binding to the distal ligand-binding site leads to an exchange of the axial ligand to a histidine, possibly H95. We show that progesterone binds to hPGRMC1 and introduces spectral changes that manifest conformational changes to the heme. Our data offer the first direct evidence supporting progesterone-binding activity of PGRMC1