Does the reduction of c heme trigger the conformational change of crystalline nitrite reductase?

The structures of nitrite reductase from Paracoccus denitrificans GB17 (NiR-Pd) and Pseudomonas aeruginosa (NiR-Pa) have been described for the oxidized and reduced state (Fül?p, V., Moir, J. W. B., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Nurizzo, D., Silvestrini, M. C., Mathieu, M., Cutruzzolà, F., Bourgeois, D., Fül?p, V., Hajdu, J., Brunori, M., Tegoni, M., and Cambillau, C. (1997) Structure 5, 1157-1171; Nurizzo, D., Cutruzzolà, F., Arese, M., Bourgeois, D., Brunori, M., Cambillau, C. , and Tegoni, M. (1998) Biochemistry 37, 13987-13996). Major conformational rearrangements are observed in the extreme states although they are more substantial in NiR-Pd. The four structures differ significantly in the c heme domains. Upon reduction, a His17/Met106 heme-ligand switch is observed in NiR-Pd together with concerted movements of the Tyr in the distal site of the d1 heme (Tyr10 in NiR-Pa, Tyr25 in NiR-Pd) and of a loop of the c heme domain (56-62 in NiR-Pa, 99-116 in NiR-Pd). Whether the reduction of the c heme, which undergoes the major rearrangements, is the trigger of these movements is the question addressed by our study. This conformational reorganization is not observed in the partially reduced species, in which the c heme is partially or largely (15-90%) reduced but the d1 heme is still oxidized. These results suggest that the d1 heme reduction is likely to be responsible of the movements. We speculate about the mechanistic explanation as to why the opening of the d1 heme distal pocket only occurs upon electron transfer to the d1 heme itself, to allow binding of the physiological substrate NO2- exclusively to the reduced metal center.     

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. M?ssbauer and EPR characterization of the heme groups

M?ssbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. M?ssbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the M?ssbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

Reductive activation of the heme iron–nitrosyl intermediate in the reaction mechanism of cytochrome c nitrite reductase: a theoretical study

Cytochrome c nitrite reductase catalyzes the six-electron, seven-proton reduction of nitrite to ammonia without release of any detectable reaction intermediate. This implies a unique flexibility of the active site combined with a finely tuned proton and electron delivery system. In the present work, we employed density functional theory to study the recharging of the active site with protons and electrons through the series of reaction intermediates based on nitrogen monoxide [Fe(II)-NO(+), Fe(II)-NO·, Fe(II)-NO(-), and Fe(II)-HNO]. The activation barriers for the various proton and electron transfer steps were estimated in the framework of Marcus theory. Using the barriers obtained, we simulated the kinetics of the reduction process. We found that the complex recharging process can be accomplished in two possible ways: either through two consecutive proton-coupled electron transfers (PCETs) or in the form of three consecutive elementary steps involving reduction, PCET, and protonation. Kinetic simulations revealed the recharging through two PCETs to be a means of overcoming the predicted deep energetic minimum that is calculated to occur at the stage of the Fe(II)-NO· intermediate. The radical transfer role for the active-site Tyr(218), as proposed in the literature, cannot be confirmed on the basis of our calculations. The role of the highly conserved calcium located in the direct proximity of the active site in proton delivery has also been studied. It was found to play an important role in the substrate conversion through the facilitation of the proton transfer steps.     

On the computation of the tertiary structure of globular proteins—IV. Use of secondary structure information

The distance geometry approach for computing the tertiary structure of globular proteins emphasized in this series of papers (Goelet al., J. theor. Biol. 99, 705–757, 1982) is developed further. This development includes incorporation of some secondary structure information—the location of alpha helices in the primary sequence—in the algorithm to compute the tertiary structure of alpha helical globular proteins. An algorithm is developed which estimates the interresidue distances between chain-proximate helices. These distances, in conjunction with the global statistical average distances obtainable from a database of real proteins and determined by the primary sequence of the protein under study, are used to determine the tertiary structure. Five proteins, parvalbumin, hemerythrin, human hemoglobin, lamprey hemoglobin, and sperm whale myoglobin, are investigated. The root mean square (RMS) errors between the calculated structures and those determined by X-ray diffraction range from 4.78 to 7.56 Å. These RMSs are 0.21–2.76 Å lower than those estimated without the secondary structure information. Contact maps and three-dimensional backbone representations also show considerable improvements with the introduction of secondary structure information.     

On the structure of the human striate area. Lamina IVcβ

Summary Layer IVc of the human striate area consists mainly of a great number of small spinous local circuit neurons which store numerous characteristic lipofuscin granules. Since the neurons of the neighbouring layers are almost devoid of pigment deposits the boundaries of lamina IVc are easily traceable. Hence, the pigment granules can be used as internal markers to unequivocally identify these small pigmented spinous local circuit neurons of lamina IVc in ultrathin sections. They have a large spherical nucleus surrounded by a narrow cytoplasmic rim poor in organelles, and very scarcely receive axosomatic symmetric synapses.Within layer IVc four types of synaptic boutons can be distinguished. Type-1-boutons are large, contain a few and loosely arranged round vesicles and make asymmetric synaptic contacts with dendrites and dendritic spines. The type-2-boutons which are also large are filled with densely packed round vesicles which accumulate at the presynaptic membrane. The large type-3-boutons are characterized by elongated vesicles and symmetric synaptic contact zones. These boutons generate several fingerlike protrusions. Small profiles which contain elongated vesicles and form symmetric synaptic contacts, are most probably parts of these protrusions. The large amount of small boutons with round vesicles and asymmetric synaptic contact zones are tentatively described as type-4-boutons although it is far from certain that they represent a uniform class. The presumable origins of the different types of boutons are discussed.Supported by the Deutsche Forschungsgemeinschaft (Br. 634/1)Dedicated to Prof. Dr. med. H. Leonhardt in honor of his 60th birthday     

Electron-paramagnetic-resonance studies of the mechanism of leaf nitrite reductase. Signals from the iron–sulphur centre and haem under turnover conditions

Low-temperature e.p.r. spectra are presented of nitrite reductase purified from leaves of vegetable marrow (Cucurbita pepo). The oxidized enzyme showed a spectrum at g=6.86, 4.98 and 1.95 corresponding to high-spin Fe(3+) in sirohaem, which disappeared slowly on treatment with nitrite. The midpoint potential of the sirohaem was estimated to be -120mV. On reduction with Na(2)S(2)O(4) or Na(2)S(2)O(4)+Methyl Viologen a spectrum at g=2.038, 1.944 and 1.922 was observed, due to a reduced iron-sulphur centre. The midpoint potential of this centre was very low, about -570mV at pH8.1, decreasing with increasing pH. On addition of cyanide, which binds to haem, and Na(2)S(2)O(4), the iron-sulphur centre became further reduced. We think that this is due to an increased midpoint potential of the iron-sulphur centre. Other ligands to haem, such as CO and the reaction product NH(3), had similar but less pronounced effects, and also changed the lineshape of the iron-sulphur signal. Samples were prepared of the enzyme frozen during the reaction with nitrite, Methyl Viologen and Na(2)S(2)O(4) in various proportions. Signals were interpreted as due to the reduced iron-sulphur centre (with slightly different g values), a haem-NO complex and reduced Methyl Viologen. In the presence of an excess of nitrite, the haem-NO spectrum was more intense, whereas in the presence of an excess of Na(2)S(2)O(4) it was weaker, and disappeared at the end of the reaction. A reaction sequence is proposed for the enzyme, in which the haem-NO complex is an intermediate, followed by other e.p.r.-silent states, leading to the production of NH(4) (+).     

Do cytochromes function as oxygen sensors in the regulation of nitrate reductase biosynthesis?

The observation that oxygen represses nitrate reductase biosynthesis in a hemA mutant grown aerobically with or without delta-aminolevulinic acid indicates that cytochromes are not responsible for nitrate reductase repression in aerobically grown cells.     

Pocket analysis of the full-length cholix toxin. An assessment of the structure–dynamics of the apo catalytic domain

Cholix toxin from Vibrio cholerae is the third member of the diphtheria toxin (DT) group of mono-ADP-ribosyltransferase (mART) bacterial toxins. It shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae DT. Cholix toxin is an important model for the development of antivirulence approaches and therapeutics against these toxins from pathogenic bacteria. Herein, we have used the high-resolution X-ray structure of full-length cholix complexed with NAD⁺ to describe the properties of the NAD⁺-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD⁺ substrate interaction. The full-length apo cholix structure is used to describe the putative NAD⁺-binding site(s) and to correlate biochemical with crystallographic data to study the stoichiometry and orientation of bound NAD⁺ molecules. We quantitatively describe the NAD⁺ substrate interactions on a residue basis for the main 22 pocket residues in cholix_f, a glycerol and 5 contact water molecules as part of the recognition surface by the substrate according to the conditions of crystallization. In addition, the dynamic properties of an in silico version of the catalytic domain were investigated in order to understand the lack of electronic density for one of the main flexible loops (R-loop) in the pocket of X-ray complexes. Implications for a rational drug design approach for mART toxins are derived.     

M?ssbauer spectroscopic studies of Escherichia coli sulfite reductase. Evidence for coupling between the siroheme and Fe4S4 cluster prosthetic groups

Escherichia coli NADPH-sulfite reductase is a complex hemoflavoprotein with an alpha 8 beta 4 subunit structure. The beta-subunits each contain one siroheme and a tetranuclear iron-sulfur center (Fe4S4). Isolated beta-monomers can catalyze the 6-electron reduction of sulfite to sulfide. We have studied the beta-monomers with M?ssbauer and EPR spectroscopy. The data show conclusively that the siroheme and the Fe4S4 cluster are strongly exchange-coupled. This is proven by the observations that (a) the two chromophores share a single electronic spin and (b) the addition of 1 electron to oxidized sulfite reductase changes the environments of 5 iron atoms. Spin-sharing is demonstrated in oxidized and 2-electron-reduced sulfite reductase and strongly implicated in 1-electron-reduced material. Thus, sulfite reductase provides the first example of an active site where a heme and an iron-sulfur cluster are closely linked as a functional unit, probably via a common bridging ligand.     

Genetic and karyotypic structure in the shrews of the Sorex araneus group: are they independent?

The species of the common shrew (Sorex araneus) group are morphologically very similar but exhibit high levels of karyotypic variation. Here we used genetic variation at 10 microsatellite markers in a data set of 212 individuals mostly sampled in the western Alps and composed of five karyotypic taxa (Sorex coronatus, Sorex antinorii and the S. araneus chromosome races Cordon, Bretolet and Vaud) to investigate the concordance between genetic and karyotypic structure. Bayesian analysis confirmed the taxonomic status of the three sampled species since individuals consistently grouped according to their taxonomical status. However, introgression can still be detected between S. antinorii and the race Cordon of S. araneus. This observation is consistent with the expected low karyotypic complexity of hybrids between these two taxa. Geographically based cryptic substructure was discovered within S. antinorii, a pattern consistent with the different postglaciation recolonization routes of this species. Additionally, we detected two genetic groups within S. araneus notwithstanding the presence of three chromosome races. This pattern can be explained by the probable hybrid status of the Bretolet race but also suggests a relatively low impact of chromosomal differences on genetic structure compared to historical factors. Finally, we propose that the current data set (available at http://www.unil.ch/dee/page7010_en.html#1) could be used as a reference by those wanting to identify Sorex individuals sampled in the western Alps.     

A role of VIPP1 as a dynamic structure within thylakoid centers as sites of photosystem biogenesis?

The vesicle inducing protein in plastids (VIPP1) is an essential protein for the biogenesis of thylakoids in modern cyanobacteria, algae, and plants. Although its exact function is still not clear, recent work has provided important hints to its mode of action. We believe that these data are consistent with a structural role of VIPP1 within thylakoid centers, which are considered as sites from which thylakoid membranes emerge and at which the biogenesis at least of photosystem II is thought to occur. Here we present a model that may serve as starting point for future research.     

Novel alkenal/one reductase activity of yeast NADPH:quinone reductase Zta1p. Prospect of the functional role for the ζ-crystallin family

ζ-Crystallins are a Zn(2+)-lacking enzyme group with quinone reductase activity, which belongs to the medium-chain dehydrogenase/reductase superfamily. It has been recently observed that human ζ-crystallin is capable of reducing the α,β-double bond of alkenals and alkenones. Here we report that this activity is also shared by the homologous Zta1p enzyme from Saccharomyces cerevisiae. While the two enzymes show similar substrate specificity, human ζ-crystallin exhibits higher activity with lipid peroxidation products and Zta1p is more active with cinnamaldehyde. The presence of Zta1p has an in vivo protective effect on yeast strains exposed to the toxic substrate 3-penten-2-one. Analysis of ZTA1 gene expression indicates an induction under different types of cellular stress, including ethanol and dimethylsulfoxide exposure and by reaching the stationary growth phase. The role of Zta1p in the yeast adaptation to some stress types and the general functional significance of ζ-crystallins are discussed.     

Genetic structure of an isolated group of the indigenous population of northern Siberia, the Nganasans (Tavginians) of Ta?mir

Demographic data of genetic interest were studied in presently living population in comparison with preseding generations of Nganasans. Decrease of sex ratio in the whole population has been revealed along with the reduction of reproductive and, possibly, effective size. The number and variance of livebirths per female were 7.29 and 9.86 respectively. Crow' index of the opportunity for selection (I) and its components (Im and If) were estimated. I was found to be 1.17, whereas Im and If--1.56 and 0.18 respectively. Linear pattern of settling in the past as well as the type of migration between adjoining subpopulations depended on culture and economy of arctic reindeer hunters as well as landscape character.     

Host dispersal as the driver of parasite genetic structure: a paradigm lost?

Understanding traits influencing the distribution of genetic diversity has major ecological and evolutionary implications for host–parasite interactions. The genetic structure of parasites is expected to conform to that of their hosts, because host dispersal is generally assumed to drive parasite dispersal. Here, we used a meta‐analysis to test this paradigm and determine whether traits related to host dispersal correctly predict the spatial co‐distribution of host and parasite genetic variation. We compiled data from empirical work on local adaptation and host–parasite population genetic structure from a wide range of taxonomic groups. We found that genetic differentiation was significantly lower in parasites than in hosts, suggesting that dispersal may often be higher for parasites. A significant correlation in the pairwise genetic differentiation of hosts and parasites was evident, but surprisingly weak. These results were largely explained by parasite reproductive mode, the proportion of free‐living stages in the parasite life cycle and the geographical extent of the study; variables related to host dispersal were poor predictors of genetic patterns. Our results do not dispel the paradigm that parasite population genetic structure depends on host dispersal. Rather, we highlight that alternative factors are also important in driving the co‐distribution of host and parasite genetic variation.