Crystal structures of the murine cytokine-inducible nitric oxide synthase oxygenase dimer with active-center water molecules, the substrate L-arginine (L-Arg), or product analog thiocitrulline reveal how dimerization, cofactor tetrahydrobiopterin, and L-Arg binding complete the catalytic center for synthesis of the essential biological signal and cytotoxin nitric oxide. Pterin binding refolds the central interface region, recruits new structural elements, creates a 30 angstrom deep active-center channel, and causes a 35 degrees helical tilt to expose a heme edge and the adjacent residue tryptophan-366 for likely reductase domain interactions and caveolin inhibition. Heme propionate interactions with pterin and L-Arg suggest that pterin has electronic influences on heme-bound oxygen. L-Arginine binds to glutamic acid-371 and stacks with heme in an otherwise hydrophobic pocket to aid activation of heme-bound oxygen by direct proton donation and thereby differentiate the two chemical steps of nitric oxide synthesis.

The Neurospora crassa photoreceptor Vivid tunes blue-light responses and modulates gating of the circadian clock. Crystal structures of dark-state and light-state Vivid reveal a light, oxygen, or voltage Per-Arnt-Sim domain with an unusual N-terminal cap region and a loop insertion that accommodates the flavin cofactor. Photoinduced formation of a cystein-flavin adduct drives flavin protonation to induce an N-terminal conformational change. A cysteine-to-serine substitution remote from the flavin adenine dinucleotide binding site decouples conformational switching from the flavin photocycle and prevents Vivid from sending signals in Neurospora. Key elements of this activation mechanism are conserved by other photosensors such as White Collar-1, ZEITLUPE, ENVOY, and flavin-binding, kelch repeat, F-BOX 1 (FKF1).The PAS (Per-Arnt-Sim) protein superfamily transduces signals from diverse biological cues, often by coupling cofactor chemistry to alterations in protein conformation or association (1). The canonical PAS domain protein photoactive yellow protein (PYP) and the light, oxygen, or voltage (LOV) PAS subclass sense blue light in bacteria, plants, and fungi (2, 3). Despite extensive photochemical and structural characterization of such bluelight sensors (2, 4-8), the mechanism by which cofactor excitation leads to biological signal propagation remains an open question.The filamentous fungus Neurospora crassa employs two blue-light sensors with LOV domains, White Collar-1 (WC-1) and Vivid (VVD) to regulate a variety of light responses (9). WC-1 and nonphotosensitive WC-2 form a complex (WCC) that resets the circadian clock by activating transcription of the clock oscillator protein Frequency (FRQ), as well as many other genes (9, 10). VVD, a small PAS protein devoid of auxiliary domains, tunes Neurospora's blue-light response by attenuating activation of the WCC. VVD is essential for response to changing levels of light and for adaptation under constant light (11)(12)(13)(14). VVD and WC-1 share sequence similarity in a core LOV domain and surrounding regions (15). Swapping the WC-1 core LOV domain with that from VVD maintains some light responses in Neurospora (16). VVD and WC-1 require flavin adenine dinucleotide (FAD) for activity instead of flavin mononucleotide (FMN), which is used by plant and algal LOV-containing proteins known as phototropins (9,12,17,18).We report the crystal structure of VVD in its dark-and light-adapted states and show how chemical changes at the active center generate conformational change at the N terminus of

Materials and Methods Figs. S1 to S3 Table S1 References S1 SUPPORTING MATERIAL Materials and Methods Preparation of Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu I Mutant azurins were expressed and Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu I was prepared using previously published protocols (S1,S2). Crystal Structure of Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu II Crystals of Re(4,7-dimethyl-1,10-phenanthroline)(CO) 3 (H 124){T 124 H|K 122 W|H 83 Q}(Cu II)azurin (Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu II ; space group I222, cell dimensions 63.22 × 69.08 × 68.94 Å 3 ; α = β = γ = 90.00°, one molecule per asymmetric unit) grew from 4 μL drops made from equal volumes of 30 mg/mL Re I (CO) 3 (dmp)(H 124)|(W 122)|AzCu II in 25 mM HEPES pH 7.5 and reservoir by vapor diffusion. The drops were equilibrated against 500 μL of reservoir