Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4

A fundamental aspect of cytochrome P450 function is the role of the strictly conserved axial cysteine ligand, replacement of which by histidine has invariably resulted in mammalian and bacterial preparations devoid of heme. Isolation of the His-436 variant of NH2-truncated P450 2B4 partly as the holoenzyme was achieved in the present study by mutagenesis of the I-helix Ala-298 residue to Glu and subsequent conversion of the axial Cys-436 to His. The expressed A298E/C436H double mutant, cloned with a hexahistidine tag, had a molecular mass equivalent to that of the primary structure of His-tagged truncated 2B4 and the sum of the two mutated residues, and contained a heme group which, when released on HPLC, showed a retention time and spectrum identical to those of iron protoporphyrin IX. The absolute spectra of A298E/C436H indicate a change in heme coordination structure from low- to high-spin, and, as expected for a His-ligated hemeprotein, the Soret maximum of the ferrous CO complex is at 422 nm. The double mutant has no oxygenase activity with representative substrates known to undergo transformation by the oxene [(FeO)3+] or peroxo activated oxygen species, but catalyzes significant H2O2 formation that is NADPH- and time-dependent, and directly proportional to the concentration of A298E/C436H in the presence of saturating reductase. Moreover, the catalytic efficiency of A298E/C436H in the H2O2-supported peroxidation of pyrogallol is more than two orders of magnitude greater than that of wild-type 2B4 or the A298E variant. The results unambiguously demonstrate that the proximal thiolate ligand is essential for substrate oxygenation by P450.     

Shape of the lateral mandibular outline in Lemuridae: a quantitative analysis of variability using elliptical Fourier analysis

While several morphometric analyses in lemurids have focused on the craniofacial complex, the characterization of their mandibular morphology has received less attention. The mandibular outline, in lateral perspective, was quantified using elliptical Fourier analysis, in an osteological sample encompassing 189 lemurid mandibles (66 Eulemur, 51 Hapalemur, 22 Lemur and 50 Varecia), and compared using multivariate statistical techniques. The taxonomic value of this outline in Lemuridae was demonstrated by the existence of significant separations between the four genera studied. In particular, the mandibular morphology of Hapalemur was markedly different from that in the group Eulemur-Lemur-Varecia. Excluding Hapalemur from analysis, the distinctions between Eulemur, Lemur and Varecia were enhanced suggesting the existence of more subtle intergeneric differences in mandibular morphology. Variation in mandibular form was greatest in Hapalemur and smallest in Eulemur and Varecia (as demonstrated by the mean values of interindividual distances); variation was higher in Lemur than in Eulemur and Varecia, but not higher than in Hapalemur. This morphological diversity may be related to functional adaptation in response to particular dietary habits. The patterns of intergeneric and intrageneric shape variations of the mandible in Lemuridae presented here provide a valuable resource for the analysis of variation among living and fossil lemurids.     

A model for the three-dimensional structure of human plasma vitronectin from small-angle scattering measurements

Small-angle X-ray scattering (SAXS) measurements were used to characterize vitronectin, a circulatory protein found in human plasma that functions in regulating cell adhesion and migration, as well as proteolytic cascades that affect blood coagulation, fibrinolysis, and pericellular proteolysis. SAXS measurements were taken over a 3-fold range of protein concentrations, yielding data that characterize a monodisperse system of particles with an average radius of gyration of 30.3 +/- 0.6 A and a maximum linear dimension of 110 A. Shape restoration was applied to the data to produce two models of the solution structure of the ligand-free protein. A low-resolution model of the protein was generated that indicates the protein to be roughly peanut-shaped. A better understanding of the domain structure of vitronectin resulted from low-resolution models developed from available high-resolution structures of the domains. These domains include the N-terminal domain that was determined experimentally by NMR [Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366] and the docked structure of the central and C-terminal domains that were determined by computational threading [Xu, D., Baburaj, K., Peterson, C. B., and Xu, Y. (2001) Proteins: Struct., Funct., Genet. 44, 312-320]. This model provides an indication of the disposition of the central domain and C-terminal heparin-binding domains of vitronectin with respect to the N-terminal somatomedin B (SMB) domain. This model constructed from the available domain structures, which agrees with the low-resolution model produced from the SAXS data, shows the SMB domain well separated from the central and heparin-binding domains by a disordered linker (residues 54-130). Also, binding sites within the SMB domain are predicted to be well exposed to the surrounding solvent for ease of access to its various ligands.     

Three-dimensional structure of the KChIP1-Kv4.3 T1 complex reveals a cross-shaped octamer

Brain I(A) and cardiac I(to) currents arise from complexes containing Kv4 voltage-gated potassium channels and cytoplasmic calcium-sensor proteins (KChIPs). Here, we present X-ray crystallographic and small-angle X-ray scattering data that show that the KChIP1-Kv4.3 N-terminal cytoplasmic domain complex is a cross-shaped octamer bearing two principal interaction sites. Site 1 comprises interactions between a unique Kv4 channel N-terminal hydrophobic segment and a hydrophobic pocket formed by displacement of the KChIP H10 helix. Site 2 comprises interactions between a T1 assembly domain loop and the KChIP H2 helix. Functional and biochemical studies indicate that site 1 influences channel trafficking, whereas site 2 affects channel gating, and that calcium binding is intimately linked to KChIP folding and complex formation. Together, the data resolve how Kv4 channels and KChIPs interact and provide a framework for understanding how KChIPs modulate Kv4 function.     

A sensitive channel family replete with sense and motion

Two new studies demonstrate that transmembrane voltage-sensing domains can act without an obvious pore-forming domain to sense membrane potential changes and make a proton-selective pore.     

Kinetic, spectroscopic, and structural investigations of the soybean lipoxygenase-1 first-coordination sphere mutant, Asn694Gly

In wild-type soybean LO-1 (WT sLO-1), Asn694 is a weak sixth ligand that is thought to be critical for enzymatic catalysis. In this investigation, N694G sLO-1 was studied to probe its contribution at this sixth ligand position to the kinetic and spectroscopic properties. The k(cat) value of N694G is approximately 230 times lower than that of WT sLO-1 at 25 degrees C, which can be partially explained by a lowered reduction potential of the iron as seen as a shift in the visible ligand-to-metal charge-transfer band (lambda(max) = 410 nm for N694G and lambda(max) = 425 nm for WT sLO-1). This conclusion was supported by a faster rate of oxidation of N694G by the product than that of WT sLO-1 (k(2) = 606 s(-)(1) for N694G and k(2) = 349 s(-)(1) for WT sLO-1). These results suggest a stronger ligand at the active site iron than the native Asn694, which is confirmed to be a water bound to the Fe(II) in the crystal structure. This produces a six-coordinate circular dichroism/magnetic circular dichroism (CD/MCD) spectra for ferrous N694G and an intermediate rhombic electron paramagnetic resonance (EPR) signal for ferric N694G. The EPR spectrum and its pH dependence suggest that the coordination environment of ferric N694G contains one hydroxide and one water. On the basis of both kinetic and structural factors, we propose that the Asn694 water-derived ligand would likely be a hydroxide and the active site, water-derived ligand a water in the ferric state, hence lowering the reaction rate of N694G more than would be expected from the lowered reduction potential alone.     

Genetics of smoking and depression

Smoking and depression are significant public health problems with multiple etiological dimensions and outcomes. Although each condition is important by itself, they are important because they often potentiate each other. Consequently, it is also essential to understand the nature their relationship. This representative review focuses on the genetic etiology of the relationship in the context of reviewing first the epidemiology of depression and smoking, and then by exploring behavioral and molecular genetic studies, and other psychiatric and medical comorbidities. At this point, epidemiological evidence for a relationship between depression and smoking/nicotine dependence is compelling. Although behavioral genetic results differ somewhat by gender and in accordance with specific definitions of depression and smoking variables, recent studies show converging evidence for common genetic factors underlying the relationship, often in addition to non-shared environmental factors. The search for underlying genes and genetic mechanisms is at an early stage, but shows promising candidate genes and genetic approaches for future studies.     

Hypothermic reconditioning by gaseous oxygen persufflation after cold storage of porcine kidneys

BackgroundDelayed graft function still represents a major complication in clinical kidney transplantation. Here we tested the possibility to improve functional outcome of cold stored kidneys a posteriori by hypothermic reconditioning using retrograde oxygen persufflation (ROP) immediately prior to reperfusion.MethodsKidneys from female German Landrace pigs were flushed with Histidine–Tryptophan–Ketoglutarate (HTK) solution and cold-stored for 18 h (control).Some grafts were subsequently subjected to 90 min of retrograde oxygen persufflation (ROP) via the renal vein during cold preservation. Early graft function of all kidneys was assessed thereafter by warm reperfusion in vitro (n = 6, resp.).ResultsRenal function upon reperfusion was significantly enhanced by ROP with an approximately twofold increase in renal clearances of creatinine and urea. ROP also led to higher renal vascular flow rates, enhanced urine output and mitigated histological alterations.ConclusionIt is concluded that initial graft function can be improved by 90 min of hypothermic gaseous oxygenation after arrival of the preserved organ in the transplantation clinic.     

Effects of multiple matings on reproductive fitness of male and female Diaeretiella rapae

Mating frequency and the amount of sperm transferred during mating have important consequences on progeny sex ratio and fitness of haplodiploid insects. Production of female offspring may be limited by the availability of sperm for fertilizing eggs. This study examined multiple mating and its effect on fitness of the cabbage aphid parasitoid Diaeretiella rapae McIntosh (Hymenoptera: Aphidiidae). Female D. rapae mated once, whereas males mated with on average more than three females in a single day. The minimum time lag between two consecutive matings by a male was 3 min, and the maximum number of matings a male achieved in a day was eight. Sperm depletion occurred as a consequence of multiple mating in D. rapae. The number of daughters produced by females that mated with multiple‐mated males was negatively correlated with the number of matings achieved by these males. Similarly, the proportion of female progeny decreased in females that mated with males that had already mated three times. Although the proportion of female progeny resulting from multiple mating decreased, the decrease was quicker when the mating occurred on the same day than when the matings occurred once per day over several days. Mating success of males initially increased after the first mating, but then males became ‘exhausted’ in later matings; their mating success decreased with the number of prior matings. The fertility of females was affected by mating with multiple‐mated males. The study suggests that male mating history affects the fitness of male and female D. rapae.