High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged 〈Azz〉 element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.

Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2–4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-l-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase—programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.

Relationship between the oxidation potential and electron spin density of the primary electron donor in reaction centers from Rhodobacter sphaeroides

The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll a molecules, labeled L or M based on their proximity to the symmetry-related protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll–protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to ≈60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron–nuclear–nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from ≈2:1 to ≈5:1 in the mutants compared with ≈2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation–reduction midpoint potential.