Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are ...indispensable for the efficient structure‐based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N‐abietoyl‐l‐tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild‐type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near‐atomic resolution of 1.22 Å. Using these crystals as seeds in a cross‐microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem‐domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.
Crystal rain: Using microcrystals of CYP102A1 (P450BM3) bound to the crystallisation accelerator N‐abietoyl‐l‐tryptophan (AbiATrp) as nuclei in batch microseeding, a range of novel structures of the P450BM3 haem domain could be determined. These structures could be useful in the structure‐based design of P450BM3 as a potent biocatalyst.
Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N₂O). Here, we report the crystal ...structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (FeB) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.
The heme importer from pathogenic bacteria is a member of the ATP-binding cassette (ABC) transporter family, which uses the energy of ATP-binding and hydrolysis for extensive conformational changes. ...Previous studies have indicated that conformational changes after heme translocation are triggered by ATP-binding to nucleotide binding domains (NBDs) and then, in turn, induce conformational transitions of the transmembrane domains (TMDs). In this study, we applied a template-based iterative all-atom molecular dynamics (MD) simulation to predict the ATP-bound outward-facing conformation of the Burkholderia cenocepacia heme importer BhuUV-T. The resulting model showed a stable conformation of the TMD with the cytoplasmic gate in the closed state and the periplasmic gate in the open state. Furthermore, targeted MD simulation predicted the intermediate structure of an occluded form (Occ) with bound ATP, in which both ends of the heme translocation channel are closed. The MD simulation of the predicted Occ revealed that Ser147 on the ABC signature motifs (LSGGQ/E) of NBDs occasionally flips and loses the active conformation required for ATP-hydrolysis. The flipping motion was found to be coupled to the inter-NBD distance. Our results highlight the functional significance of the signature motif of ABC transporters in regulation of ATPase and chemo-mechanical coupling mechanism.
The crystal structures of bacterial nitric oxide reductases (NOR) from Pseudomonas aeruginosa and Geobacillus stearothermophilus were reported. The structural characteristics of these enzymes, ...especially at the catalytic site and on the pathway that catalytic protons are delivered, are compared, and the corresponding regions of aerobic and micro-aerobic cytochrome oxidases, O2 reducing enzymes, which are evolutionarily related to NOR are discussed. On the basis of these structural comparisons, a mechanism for the reduction of NO to produce N2O by NOR, and the possible molecular evolution of the proton pumping ability of the respiratory enzymes is discussed. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
► Overall structures are basically similar between two NORs, cNOR and qNOR. ► Direction for the catalytic proton transfer is entirely different. ► Based on structural comparison, a possible NO reduction mechanism can be proposed.
Bacterial pathogens acquire heme from the host hemoglobin as an iron nutrient for their virulence and proliferation in blood. Concurrently, they encounter cytotoxic-free heme that escapes the ...heme-acquisition process. To overcome this toxicity, many gram-positive bacteria employ an ATP-binding cassette heme-dedicated efflux pump, HrtBA in the cytoplasmic membranes. Although genetic analyses have suggested that HrtBA expels heme from the bacterial membranes, the molecular mechanism of heme efflux remains elusive due to the lack of protein studies. Here, we show the biochemical properties and crystal structures of
HrtBA, alone and in complex with heme or an ATP analog, and we reveal how HrtBA extracts heme from the membrane and releases it. HrtBA consists of two cytoplasmic HrtA ATPase subunits and two transmembrane HrtB permease subunits. A heme-binding site is formed in the HrtB dimer and is laterally accessible to heme in the outer leaflet of the membrane. The heme-binding site captures heme from the membrane using a glutamate residue of either subunit as an axial ligand and sequesters the heme within the rearranged transmembrane helix bundle. By ATP-driven HrtA dimerization, the heme-binding site is squeezed to extrude the bound heme. The mechanism sheds light on the detoxification of membrane-bound heme in this bacterium.
Cytochrome P450 peroxygenases belonging to the CYP152 family catalyse the oxidation of fatty acids using H 2 O 2 . CYP152N1 isolated from Exiguobacterium sp. AT1b exclusively catalyses the ...α-selective hydroxylation of myristic acid at physiological H 2 O 2 concentration. However, a series of shorter-alkyl-chain fatty acids such as tridecanoic acid were produced from myristic acid by increasing the concentration of H 2 O 2 (1–10 mM). The yield of tridecanoic acid from myristic acid reached 17%. An 18 O-labeled oxidant study suggested that CYP152N1 catalysed the overoxidation of α-hydroxymyristic acid to form α-ketomyristic acid, which in turn was spontaneously decomposed by H 2 O 2 to yield tridecanoic acid. Crystal structure analysis of CYP152N1 revealed its high similarity to other CYP152 family enzymes, such as CYP152A1 and CYP152B1. MD simulations of α-hydroxymyristic acid accommodated in CYP152N1 proposed a possible pre-oxidation conformation of α-hydroxymyristic acid for the decarboxylation reaction.
Characterization of short-lived reaction intermediates is essential for elucidating the mechanism of the reaction catalyzed by metalloenzymes. Here, we demonstrated that the photolysis of a caged ...compound under cryogenic temperature followed by thermal annealing is an invaluable technique for trapping of short-lived reaction intermediates of metalloenzymes through the study of membrane-integrated nitric oxide reductase (NOR) that catalyzes reductive coupling of two NO molecules to N2O at its heme/nonheme FeB binuclear center. Although NO produced by the photolysis of caged NO did not react with NOR under cryogenic temperature, annealing to ∼160 K allowed NO to diffuse and react with NOR, which was evident from the appearance of EPR signals assignable to the S = 3/2 state. This indicates that the nonheme FeB-NO species can be trapped as the intermediate. Time-resolved IR spectroscopy with the use of the photolysis of caged NO as a reaction trigger showed that the intermediate formed at 10 μs gave the NO stretching frequency at 1683 cm–1 typical of nonheme Fe-NO, confirming that the combination of the cryo-photolysis of caged NO and annealing enabled us to trap the reaction intermediate. Thus, the cryo-photolysis of the caged compound has great potential for the characterization of short-lived reaction intermediates.
Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus ...megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.
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