Molecular mechanisms underlying the repair of nitrosylated [Fe-S] clusters by the microbial protein YtfE remain poorly understood. The X-ray crystal structure of YtfE, in combination with EPR, magnetic circular dichroism (MCD), UV, and (17) O-labeling electron spin echo envelope modulation measurements, show that each iron of the oxo-bridged Fe(II) -Fe(III) diiron core is coordinatively unsaturated with each iron bound to two bridging carboxylates and two terminal histidines in addition to an oxo-bridge. Structural analysis reveals that there are two solvent-accessible tunnels, both of which converge to the diiron center and are critical for capturing substrates. The reactivity of the reduced-form Fe(II) -Fe(II) YtfE toward nitric oxide demonstrates that the prerequisite for N2 O production requires the two iron sites to be nitrosylated simultaneously. Specifically, the nitrosylation of the two iron sites prior to their reductive coupling to produce N2 O is cooperative. This result suggests that, in addition to any repair of iron centers (RIC) activity, YtfE acts as an NO-trapping scavenger to promote the NO to N2 O transformation under low NO flux, which precedes nitrosative stress.
Bubbling O into a THF solution of Co(BDPP) (1) at -90 °C generates an O adduct, Co(BDPP)(O) (3). The resonance Raman and EPR investigations reveal that 3 contains a low spin cobalt(III) ion bound to a superoxo ligand. Significantly, at -90 °C, 3 can react with 2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOH) to form a structurally characterized cobalt(III)-hydroperoxo complex, Co(BDPP)(OOH) (4) and TEMPO. Our findings show that cobalt(III)-superoxo species are capable of performing hydrogen atom abstraction processes. Such a stepwise O-activating process helps to rationalize cobalt-catalyzed aerobic oxidations and sheds light on the possible mechanism of action for Co-bleomycin.
Proapoptotic BAX protein is largely cytosolic in healthy cells, but it oligomerizes and translocates to mitochondria upon receiving apoptotic stimuli. A long-standing challenge has been the inability to capture any structural information beyond the onset of activation. Here, we present solution structures of an activated BAX oligomer by means of spectroscopic and scattering methods, providing details about the monomer-monomer interfaces in the oligomer and how the oligomer is assembled from homodimers. We show that this soluble oligomer undergoes a direct conversion into membrane-inserted oligomer, which has the ability of inducing apoptosis and structurally resembles a membrane-embedded oligomer formed from BAX monomers in lipid environment. Structural differences between the soluble and the membrane-inserted oligomers are manifested in the C-terminal helices. Our data suggest an alternative pathway of apoptosis in which BAX oligomer formation occurs prior to membrane insertion.
In nano-confinements, aqueous solutions can be found to remain in a liquid state at subfreezing temperatures. The finding provides a means of entering into previously inaccessible temperature regions for studying the dynamics and structure of bulk liquid. Here we show that studying biomolecular structures in nano-confinements improves the accuracy of cryostructures and provides better insight into the relationship between hydration water and biomolecules. Synthetic prion protein peptides are studied in two experimental conditions: (i) in confined nanochannels within mesoporous materials, and (ii) in vitrified bulk solvents, with a temperature range of 50-275 K, using cw/pulse ESR techniques. A large inhomogeneous lineshape broadening is only observed for the spectra from the vitrified bulk solvent below 70 K, suggesting a possible peptide clustering in the solution. The spin-counting and distance measurements by DEER-ESR provide further evidence that peptides are dispersed homogeneously in mesopores but heterogeneously in vitrified solvents wherein the biomolecular structure is disturbed due to heterogeneity in the bulk solvent structure. Our study demonstrates that the nanospace within mesoporous materials provides an amorphous environment that is better than vitrified bulk solvent for studying biostructures at cryogenic temperatures.
BCL-2-associated X (BAX) protein acts as a gatekeeper in regulating mitochondria-dependent apoptosis. Under cellular stress, BAX becomes activated and transforms into a lethal oligomer that causes mitochondrial outer membrane permeabilization (MOMP). Previous studies have identified several structural features of the membrane-associated BAX oligomer; they include the formation of the BH3-in-groove dimer, the collapse of the helical hairpin α5-α6, and the membrane insertion of α9 helix. However, it remains unclear as to the role of lipid environment in determining the conformation and the pore-forming activity of the BAX oligomers. Here we study molecular details of the membrane-associated BAX in various lipid environments using fluorescence and ESR techniques. We identify the inactive versus active forms of membrane-associated BAX, only the latter of which can induce stable and large membrane pores that are sufficient in size to pass apoptogenic factors. We reveal that the presence of CL is crucial to promoting the association between BAX dimers, hence the active oligomers. Without the presence of CL, BAX dimers assemble into an inactive oligomer that lacks the ability to form stable pores in the membrane. This study suggests an important role of CL in determining the formation of active BAX oligomers.
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