The genes of N. pharaonis SRII and the carboxy terminal truncated transducer (1-114) were cloned into a pET27bmod expression vector 24 with a C-terminal £ 7 His tag, respectively. Proteins were expressed in Escherichia coli strain BL21 (DE3), and purified as described 25,26. After removal of imidazol by diethyl-aminoethyl chromatography, SRII-His and HtrII 114-His were mixed in a 1:1 ratio, followed by reconstitution into purple membrane (the bacteriorhodopsin containing membrane patches of H. salinarum) lipids 7 (protein to lipid ratio 1:35). After filtration, the reconstituted proteins were pelleted by centrifugation at 100,000g. For resolubilization, the samples were resuspended in a buffer containing 2% n-octyl-b-D-glucopyranoside and shaken for 16 h at 4 8C in the dark. The resolubilized complex was isolated by centrifugation at 100,000g. Crystallization, structure determination and refinement We added the solubilized complex in crystallization buffer (150 mM NaCl, 25 mM Na/KPi, pH 5.1, 0.8% n-octyl-b-D-glucopyranoside) to the lipidic phase, formed from monovaccenin (Nu-Chek Prep). Precipitant was 1 M salt Na/KPi, pH 5.6. Crystals were grown at 22 8C. X-ray diffraction data were collected at beamline ID14-1 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France, using a Quantum ADSC Q4R CCD (charge-coupled device) detector. Data were integrated using MOSFILM 27 and SCALA 28. Molecular replacement using MOLREP 28 to phase a polyalanine model (from Protein Data Bank accession number 1JGJ (ref. 12)) gave a unique solution (R ¼ 0.568, correlation coefficient C ¼ 0.357) at 2.9 A ˚. After inserting side chains for SRII, the helices of HtrII were found (R ¼ 0.329, C ¼ 0.711). Simulated annealing, positional refinement and temperature factor refinement were performed in CNS 29 ; model rebuilding was carried out in O 30 (Table 1).

The nitrite anion is reduced to nitric oxide (NO • ) as oxygen tension decreases. Whereas this pathway modulates hypoxic NO • signaling and mitochondrial respiration and limits myocardial infarction in mammalian species, the pathways to nitrite bioactivation remain uncertain. Studies suggest that hemoglobin and myoglobin may subserve a fundamental physiological function as hypoxia dependent nitrite reductases. Using myoglobin wild-type ( +/+ ) and knockout ( −/− ) mice, we here test the central role of myoglobin as a functional nitrite reductase that regulates hypoxic NO • generation, controls cellular respiration, and therefore confirms a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. We find that myoglobin is responsible for nitrite-dependent NO • generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO • by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In isolated myoglobin +/+ but not in myoglobin −/− hearts, nitrite treatment resulted in an improved recovery of postischemic left ventricular developed pressure of 29%. In vivo administration of nitrite reduced myocardial infarction by 61% in myoglobin +/+ mice, whereas in myoglobin −/− mice nitrite had no protective effects. These data support an emerging paradigm that myoglobin and the heme globin family subserve a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation. myoglobin knockout mice

Electron paramagnetic resonance-based inter-residue distance measurements between site-directed spinlabelled sites of sensory rhodopsin II (NpSRII) and its transducer NpHtrII from Natronobacterium pharaonis revealed a 2:2 complex with 2-fold symmetry. The core of the complex is formed by the four transmembrane helices of a transducer dimer. Upon light excitation, the previously reported¯ap-like movement of helix F of NpSRII induces a conformational change in the transmembrane domain of the transducer. The inter-residue distance changes determined provide strong evidence for a rotary motion of the second transmembrane helix of the transducer. This helix rotation becomes uncoupled from changes in the receptor during the last step of the photocycle.

The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behaviour of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance and X-ray structure analysis. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. The formation of late M has been correlated with the formation of the signalling state. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The transducer responds to the activation of the receptor by a clockwise rotation of about 15 degrees of helix TM2 and a displacement of this helix by 0.9 A at the cytoplasmic surface.