A dedicated haem lyase is required for the maturation of a novel bacterial cytochrome c with unconventional covalent haem binding

In bacterial c-type cytochromes, the haem cofactor is covalently attached via two cysteine residues organized in a haem c-binding motif. Here, a novel octa-haem c protein, MccA, is described that contains only seven conventional haem c-binding motifs (CXXCH), in addition to several single cysteine residues and a conserved CH signature. Mass spectrometric analysis of purified MccA from Wolinella succinogenes suggests that two of the single cysteine residues are actually part of an unprecedented CX15CH sequence involved in haem c binding. Spectroscopic characterization of MccA identified an unusual high-potential haem c with a red-shifted absorption maximum, not unlike that of certain eukaryotic cytochromes c that exceptionally bind haem via only one thioether bridge. A haem lyase gene was found to be specifically required for the maturation of MccA in W. succinogenes. Equivalent haem lyase-encoding genes belonging to either the bacterial cytochrome c biogenesis system I or II are present in the vicinity of every known mccA gene suggesting a dedicated cytochrome c maturation pathway. The results necessitate reconsideration of computer-based prediction of putative haem c-binding motifs in bacterial proteomes

Structure of the human κ-opioid receptor in complex with JDTic

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and—in the case of κ-opioid receptor (κ-OR)—dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5′-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure–activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR

Recent progress on obtaining theoretical and experimental support for the "E-pathway hypothesis" of coupled transmembrane electron and proton transfer in dihaem-containing quinol:fumarate reductase

econciliation of apparently contradictory experimental results obtained on the quinol: fumarate reductase (QFR), a dihaem-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called E-pathway hypothesis. According to this hypothesis, transmembrane electron transfer via the haem groups is strictly coupled to co-transfer of protons via a transiently established, novel pathway, proposed to contain the side chain of residue Glu-C180 and the distal haem ring-C propionate as the most prominent components. This hypothesis has recently been supported by both theoretical and experimental results. Multiconformation continuum electrostatics calculations predict Glu-C180 to undergo a combination of proton uptake and conformational change upon haem reduction. Strong experimental support for the proposed role of Glu-C180 in the context of the “E-pathway hypothesis” is provided by the effects of replacing Glu-C180 with Gln or Ile by site-directed mutagenesis, the consequences of these mutations for the viability of the resulting mutants, together with the structural and functional characterisation of the corresponding variant enzymes, and the comparison of redox-induced Fourier-transform infrared (FTIR) difference spectra for the wild type and Glu-C180 → Gln variant. A possible haem propionate involvement has recently been supported by combining 13C-haem propionate labelling with redox-induced FTIR difference spectroscopy

Probing Heme Propionate Involvement in Transmembrane Proton Transfer Coupled to Electron Transfer in Dihemic Quinol: Fumarate Reductase by ¹³C-Labeling and FTIR Difference Spectroscopy

Quinol:fumarate reductase (QFR) is the terminal enzyme of anaerobic fumarate respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. Although the diheme-containing QFR from Wolinella succinogenes is known to catalyze an electroneutral process, its three-dimensional structure at 2.2 Å resolution and the structural and functional characterization of variant enzymes revealed locations of the active sites that indicated electrogenic catalysis. A solution to this apparent controversy was proposed with the so-called “E-pathway hypothesis”. According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180 and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role of the former constituent. Here, we investigate a possible heme−propionate involvement in redox-coupled proton transfer by a combination of specific 13C-heme propionate labeling and Fourier transform infrared (FTIR) difference spectroscopy. The labeling was achieved by creating a W. succinogenes mutant that was auxotrophic for the heme−precursor 5-aminolevulinate and by providing [1-13C]-5-aminolevulinate to the medium. FTIR difference spectroscopy revealed a variation on characteristic heme propionate vibrations in the mid-infrared range upon redox changes of the distal heme. These results support a functional role of the distal heme ring C propionate in the context of the proposed E-pathway hypothesis of coupled transmembrane electron and proton transfe

Probing heme propionate involvement in transmembrane proton transfer coupled to electron transfer in dihemic quinol:fumarate reductase by 13C-labeling and FTIR difference spectroscopy.

Quinol:fumarate reductase (QFR) is the terminal enzyme of anaerobic fumarate respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. Although the diheme-containing QFR from Wolinella succinogenes is known to catalyze an electroneutral process, its three-dimensional structure at 2.2 A resolution and the structural and functional characterization of variant enzymes revealed locations of the active sites that indicated electrogenic catalysis. A solution to this apparent controversy was proposed with the so-called "E-pathway hypothesis". According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180 and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role of the former constituent. Here, we investigate a possible heme-propionate involvement in redox-coupled proton transfer by a combination of specific (13)C-heme propionate labeling and Fourier transform infrared (FTIR) difference spectroscopy. The labeling was achieved by creating a W. succinogenes mutant that was auxotrophic for the heme-precursor 5-aminolevulinate and by providing [1-(13)C]-5-aminolevulinate to the medium. FTIR difference spectroscopy revealed a variation on characteristic heme propionate vibrations in the mid-infrared range upon redox changes of the distal heme. These results support a functional role of the distal heme ring C propionate in the context of the proposed E-pathway hypothesis of coupled transmembrane electron and proton transfer

Chemical Determinants of Fatty Acid Amide Hydrolase Inactivation Explored by QM/MM Mechanistic Modelling

XX National Meeting on Medicinal Chemistry Book of Abstract SC21, 79

SAR and LC/MS studies of β-lactamic inhibitors of human fatty acid amide hydrolase (hFAAH) : evidence of a nonhydrolytic process

The endocannabinoid hydrolyzing enzyme FAAH uses a nonclassical catalytic triad (namely, Ser-Ser-Lys instead of Ser-Asp-His) to cleave its endogenous substrates. Because inhibiting FAAH has a clear therapeutic potential, we previously developed β-lactam-type inhibitors of hFAAH. Here, we report the synthesis of five novel derivatives (5-9) of our lead compound 1-(pent-4-enoyl)-3(S)-[1(R)-(4-phenylbutanoyloxy)-ethyl]-azetidin-2-one (4, IC(50) = 5 nM) obtained via the systematic replacement of one to three carbonyls by methylene groups. The SAR results showed that the imide, but not the lactam, function is essential to the inhibition of hFAAH. We also performed LC/MS analysis following incubation of our inhibitors with hFAAH or mouse liver. We demonstrated that hFAAH interacts with these β-lactam-type inhibitors but, unexpectedly, does not open the β-lactam moiety. This mechanism seems to be unique to FAAH because the β-lactam function of the inhibitors is hydrolyzed when they are incubated in the presence of the serine hydrolases expressed in the mouse liver. Finally, we confirmed these results by showing that a highly selective FAAH inhibitor (PF-750) does not prevent this hydrolysis by liver homogenates