Soil-borne mutualistic fungi, such as the ectomycorrhizal fungi, have helped shape forest communities worldwide over the last 180 million years through a mutualistic relationship with tree roots in which the fungal partner provides a large array of nutrients to the plant host in return for photosynthetically derived sugars. This exchange is essential for continued growth and productivity of forest trees, especially in nutrient-poor soils. To date, the signals from the two partners that mediate this symbiosis have remained uncharacterized. Here we demonstrate that MYCORRHIZAL iNDUCED SMALL SECRETED PROTEIN 7 (MiSSP7), the most highly symbiosis-upregulated gene from the ectomycorrhizal fungus Laccaria bicolor, encodes an effector protein indispensible for the establishment of mutualism. MiSSP7 is secreted by the fungus upon receipt of diffusible signals from plant roots, imported into the plant cell via phosphatidylinositol 3-phosphate-mediated endocytosis, and targeted to the plant nucleus where it alters the transcriptome of the plant cell. L. bicolor transformants with reduced expression of MiSSP7 do not enter into symbiosis with poplar roots. MiSSP7 resembles effectors of pathogenic fungi, nematodes, and bacteria that are similarly targeted to the plant nucleus to promote colonization of the plant tissues and thus can be considered a mutualism effector.

Kinetic, EPR, and Fourier transform infrared spectroscopic analysis of Desulfovibrio fructosovorans [NiFe]hydrogenase mutants targeted to Glu-25 indicated that this amino acid participates in proton transfer between the active site and the protein surface during the catalytic cycle. Replacement of that glutamic residue by a glutamine did not modify the spectroscopic properties of the enzyme but cancelled the catalytic activity except the para-H 2 /ortho-H 2 conversion. This mutation impaired the fast proton transfer from the active site that allows high turnover numbers for the oxidation of hydrogen. Replacement of the glutamic residue by the shorter aspartic acid slowed down this proton transfer, causing a significant decrease of H 2 oxidation and hydrogen isotope exchange activities, but did not change the para-H 2 /ortho-H 2 conversion activity. The spectroscopic properties of this mutant were totally different, especially in the reduced state in which a non-photosensitive nickel EPR spectrum was obtained.Many microorganisms use molecular hydrogen in their metabolic routes as an energy source or for evacuating an excess of electrons. The enzymes that catalyze reversibly the conversion of molecular hydrogen to two electrons and two protons are known as hydrogenases. Although this is the simplest chemical reaction, the catalytic mechanism of these enzymes is quite complicated, and its details are still a matter of debate (1). Hydrogen isotope exchange experiments indicate that the H 2 cleavage reaction is heterolytic; thus, a hydride and a proton are formed in the first step (2, 3). In the second step, the two electrons of the hydride are extracted, and a second proton is formed. Subsequently, the two electrons have to be transported, via the intramolecular electron transfer chain, from the active site to the redox partner of the hydrogenase (a redox protein or NAD ϩ (P)) in vivo, or a redox dye in vitro; the two protons have to be transferred to the protein environment as well. These steps are reversed in the case of H 2 production activity (1).How do all these steps take place in hydrogenases? These proteins are metalloenzymes that all contain iron, and in many cases, also nickel. The crystallographic structures of several [Fe] hydrogenases (4, 5) and [NiFe] hydrogenases (6, 7) have been obtained by x-ray diffraction studies. In both types of enzymes, the active site is a deeply buried bimetallic center, in which the metals are bridged by thiol groups and have CO and CN Ϫ as ligands. This type of coordination favors the binding of molecular hydrogen or hydride to the active site (1, 8). The crystal structures also indicate that Fe-S clusters are located between the active site and the protein surface, which are thought to form the intramolecular electron pathway in the H 2 production/oxidation mechanism (9). In [NiFe] hydrogenases, one nickel and one iron atom form the bimetallic center. The nickel is coordinated to four cysteine ligands via their thiol groups. Two of them are terminal ligands, and the other...

In light of recent experiments suggesting high-spin (HS) Ni(II) species in the catalytic cycle of [NiFe] hydrogenase, a series of models of the Ni(II) forms Ni-SI(I,II), SI-CO and Ni-R(I,II,III) were examined in their high-spin states via density functional calculations. Because of its importance in the catalytic cycle, the Ni-C form was also included in this study. Unlike the Ni(II) forms in previous studies, in which a low-spin (LS) state was assumed and a square-planar structure found, the optimized geometries of these HS Ni(II) forms resemble those observed in the crystal structures: a distorted tetrahedral to distorted pyramidal coordination for the NiS4. This resemblance is particularly significant because the LS state is 20-30 kcal/mol less stable than the HS state for the geometry of the crystal structure. If these Ni(II) forms in the enzyme are not high spin, a large change in geometry at the active site is required during the catalytic cycle. Furthermore, only the HS state for the CO-inhibited form SI-CO has CO stretching frequencies that match the experimental results. As in the previous work, these new results show that the heterolytic cleavage reaction of dihydrogen (where H2 is cleaved with the metal acting as a hydride acceptor and a cysteine as the proton acceptor) has a lower energy barrier and is more exothermic when the active site is oxidized to Ni(III). The enzyme models described here are supported by a calibrated correlation of the calculated and measured CO stretching frequencies of the forms of the enzyme. The correlation coefficient for the final set of models of the forms of [NiFe] hydrogenase is 0.8.

The kinetics of the activation and anaerobic inactivation processes of Desulfovibrio gigas hydrogenase have been measured in D(2)O by FTIR spectroelectrochemistry. A primary kinetic solvent isotope effect was observed for the inactivation process but not for the activation step. The kinetics of these processes have been also measured after replacement of a glutamic residue placed near the active site of an analogous [NiFe] hydrogenase from Desulfovibrio fructosovorans. Its replacement by a glutamine affected greatly the kinetics of the inactivation process but only slightly the activation process. The interpretation of the experimental results is that the rate-limiting step for anaerobic inactivation is the formation from water of a micro-OH(-) bridge at the hydrogenase active site, and that Glu25 has a role in this step.