Structure and function of the Gondwanian hemoglobin of Pseudaphritis urvillii, a primitive notothenioid fish of temperate latitudes

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.     

Statistical independence and neural computation in the leech ganglion

 In this report, the input/output relations in an isolated ganglion of the leech Hirudo medicinalis were studied by simultaneously using six or eight suction pipettes and two intracellular electrodes. Sensory input was mimicked by eliciting action potentials in mechanosensory neurons with intracellular electrodes. The integrated neural output was measured by recording extracellular voltage signals with pipettes sucking the roots and the connectives. A single evoked action potential activated electrical activity in at least a dozen different neurons, some of which were identified. This electrical activity was characterized by a high degree of temporal and spatial variability. The action potentials of coactivated neurons, i.e. activated by the same mechanosensory neuron, did not show any significant pairwise correlation. Indeed, the analysis of evoked action potentials indicates clear statistical independence among coactivated neurons, presumably originating from the independence of synaptic transmission at distinct synapses. This statistical independence may be used to increase reliability when neuronal activity is averaged or pooled. It is suggested that statistical independence among coactivated neurons may be a usual property of distributed processing of neuronal networks and a basic feature of neural computation. Received: 20 September 1999 / Accepted in revised form: 3 March 2000     

Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO₂⁻ binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO₂⁻ concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO₂−binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.     

Extended cardiolipin anchorage to cytochrome c: a model for protein–mitochondrial membrane binding

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.     

Climate warming could increase recruitment success in glacier foreland plants

Background and Aims Glacier foreland plants are highly threatened by global warming. Regeneration from seeds on deglaciated terrain will be crucial for successful migration and survival of these species, and hence a better understanding of the impacts of climate change on seedling recruitment is urgently needed to predict future plant persistence in these environments. This study presents the first field evidence of the impact of climate change on recruitment success of glacier foreland plants.Methods Seeds of eight foreland species were sown on a foreland site at 2500 m a.s.l., and at a site 400 m lower in altitude to simulate a 2·7 °C increase in mean annual temperature. Soil from the site of origin was used to reproduce the natural germination substrate. Recruitment success, temperature and water potential were monitored for 2 years. The response of seed germination to warming was further investigated in the laboratory.Key Results At the glacier foreland site, seedling emergence was low (0 to approx. 40 %) and occurred in summer in all species after seeds had experienced autumn and winter seasons. However, at the warmer site there was a shift from summer to autumn emergence in two species and a significant increase of summer emergence (13–35 % higher) in all species except two. Survival and establishment was possible for 60–75 % of autumn-emerged seedlings and was generally greater under warmer conditions. Early snowmelt in spring caused the main ecological factors enhancing the recruitment success.Conclusions The results suggest that warming will influence the recruitment of glacier foreland species primarily via the extension of the snow-free period in spring, which increases seedling establishment and results in a greater resistance to summer drought and winter extremes. The changes in recruitment success observed here imply that range shifts or changes in abundance are possible in a future warmer climate, but overall success may be dependent on interactions with shifts in other components of the plant community.     

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 ??-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.     

The role of CyaY in iron sulfur cluster assembly on the E. coli IscU scaffold protein

Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.     

The heme iron coordination of unfolded ferric and ferrous cytochrome c in neutral and acidic urea solutions. Spectroscopic and electrochemical studies

The heme iron coordination of unfolded ferric and ferrous cytochrome c in the presence of 7-9 M urea at different pH values has been probed by several spectroscopic techniques including magnetic and natural circular dichroism (CD), electrochemistry, UV-visible (UV-vis) absorption and resonance Raman (RR). In 7-9 M urea at neutral pH, ferric cytochrome c is found to be predominantly a low spin bis-His-ligated heme center. In acidic 9 M urea solutions the UV-vis and near-infrared (NIR) magnetic circular dichroism (MCD) measurements have for the first time revealed the formation of a high spin His/H(2)O complex. The pK(a) for the neutral to acidic conversion is 5.2. In 9 M urea, ferrous cytochrome c is shown to retain its native ligation structure at pH 7. Formation of a five-coordinate high spin complex in equilibrium with the native form of ferrous cytochrome c takes place below the pK(a) 4.8. The formal redox potential of the His/H(2)O complex of cytochrome c in 9 M urea at pH 3 was estimated to be -0.13 V, ca. 100 mV more positive than E degrees ' estimated for the bis-His complex of cytochrome c in urea solution at pH 7.     

Spectroscopic and interfacial properties of myoglobin/surfactant complexes

The complexes of horse myoglobin (Mb) with the anionic surfactant sodium dodecyl sulfate (SDS), and with the cationic surfactants cetyltrimethylammonium chloride (CTAC) and decyltrimethylammonium bromide (DeTAB), have been studied by a combination of surface tension measurements and optical spectroscopy, including heme absorption and aromatic amino acid fluorescence. SDS interacts in a monomeric form with Mb, which suggests the existence of a specific binding site for SDS, and induces the formation of a hexacoordinated Mb heme, possibly involving the distal histidine. Fluorescence spectra display an increase of tryptophan emission. Both effects point to an increased protein flexibility. SDS micelles induce both the appearance of two more heme species, one of which has the features of free heme, and protein unfolding. Mb/CTAC complexes display a very different behavior. CTAC monomers have no effect on the absorption spectra, and only a slight effect on the fluorescence spectra, whereas the formation of CTAC aggregates on the protein strongly affects both absorption and fluorescence. Mb/DeTAB complexes behave in a very similar way as Mb/CTAC complexes. The surface activity of the different Mb/surfactant complexes, as well as the interactions between the surfactants and Mb, are discussed on the basis of their structural properties.     

Mitochondrial pH monitored by a new engineered green fluorescent protein mutant

We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK(a) (pK(a)') around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca(2+) ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca(2+) accumulation by this organelle. Finally, we demonstrate that Ca(2+) mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.