Electron spin-lattice relaxation of the [Cu(1.5) ... Cu(1.5)] dinuclear copper center in nitrous oxide reductase.

Relaxation times have been obtained with time-domain EPR for the dinuclear mixed valence [CuA(1.5) ... CuA(1.5)[ S = 1/2 center in nitrous oxide reductase, N2OR, from Pseudomonas stutzeri, in the TN5 mutant defective in copper chromophore biosynthesis, in a synthetic mixed valence complex, and in type 1 and 2 copper complexes. Data confirmed that the intrinsic electron spin-lattice relaxation time, T1, for N2OR in the temperature range of 6-25 K is unusually short for copper centers. At best, a twofold increase of T1 from g perpendicular to g parallel was measured. Optimized fits of the saturation-recovery data were obtained using both double-exponential and stretched-exponential functions. The temperature dependence of the spin-lattice relaxation rate of mutant N2OR is about T5.0 with the stretched-exponential model or T3.3 and T3.9 for the model using the sum of two exponentials. These T1s are intrinsic to the mixed valence [CuA(1.5) ... CuA(1.5)] center, and no interaction of the second copper center in wild-type N2OR with the [CuA(1.5) ... CuA(1.5)] center has been observed. The T1 of the mixed valence center of N2OR is not only shorter than for monomeric square planar Cu(II) complexes, but also shorter than for a synthetic mixed valence complex, Cu2(N[CH2CH2NHCH2CH2NHCH2CH2]3N). The short T1 is attributed to the vibrational modes of type 1 copper and/or the metal-metal interaction in [CuA(1.5) ... CuA(1.5)].     

Area postrema and adjacent solitary nucleus in water and energy balance

Lesions of the area postrema (AP) and immediately adjacent nucleus tractus solitarii cause a syndrome of permanent weight loss with little disruption to behavioral controls of body weight regulation. There is also a permanent polydipsia and elevated salt appetite, which appear to be secondary to deficits in renal conservation of water and sodium. Neuroanatomical studies indicate that the immediately adjacent solitary nucleus and AP are sites of termination of visceral afferents from abdominal organs and the destination and origin of distant central afferent and efferent connections.     

ESR parameters for cupric bleomycin in the mobilized state

The configuration of the copper complex of the glycopeptide bleomycin, CuBlm, is presumed to be pyramidal square planar from a previous X-ray structural determination of a fragment of cupric bleomycin. This study presents evidence for a difference in the ESR parameters for cupric bleomycin in the liquid as opposed to the solid state. A decrease in Aiso for CuBlm in the liquid state can be directly surmised from the low frequency S-band spectrum for which three of the four cupric hyperfine lines are partially resolved. Computer simulated spectra infer that the absolute value of All increases about 100 MHz and the value of Al may change sign for CuBlm in the liquid state. Simulations using a rotational correlation time of about 250 psec. indicate that CuBLM may not be spherical in the liquid phase. The fastest component for anisotropic motion could dominate and account for the well resolved cupric hyperfine structure. Furthermore, it is argued from an analysis of the cupric hyperfine coupling constants that the CuBlm structure opens up at room temperature and that the cupric ion is displaced from the square plane.     

NMR studies of the activation of the Escherichia coli trp repressor

The Escherichia coli trp repressor binds to the trp operator in the presence of tryptophan, thereby inhibiting tryptophan biosynthesis. Tryptophan analogues lacking the alpha-amino group act as inducers of trp operon expression. We have used one- and two-dimensional 1H-NMR spectroscopy to compare the binding to the repressor of the corepressors L-tryptophan, D-tryptophan and 5-methyl-DL-tryptophan with that of the inducer indole-3-propionic acid. We have determined the chemical shifts of the indole ring protons of the ligands when bound to the protein, principally by magnetization-transfer experiments. The chemical shifts of the indole NH and C4 protons differ between corepressors and inducer. At the same time, the pattern of intermolecular NOE between protons of the protein and those of the ligand also differ between the two classes of ligand. These two lines of evidence indicate that corepressors and inducers bind differently in the binding site, and the evidence suggests that the orientation of the indole ring in the binding site differs by approximately 180 degrees between the two kinds of ligand. This is in contrast to a previous solution study [Lane, A.N. (1986) Eur. J. Biochem. 157, 405-413], but consistent with recent X-ray crystallographic work [Lawson, C.L. & Sigler, P.B. (1988) Nature 333, 869-871]. D-Tryptophan and 5-methyltryptophan, which are more effective corepressors than L-tryptophan, bind similarly to L-tryptophan. The indole ring of D-tryptophan appears to bind in essentially the same orientation as that of the L isomer. There are, however, some differences in chemical shifts and NOE for 5-methyltryptophan, which indicate that there are significant differences between the two corepressors L-tryptophan and 5-methyltryptophan in the orientation of the indole ring within the binding site.     

Is the mammalian cell plasma membrane a barrier to oxygen transport?

Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.     

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author