therapy may allow for greater exercise tolerance, ease the burden of exercise compliance and facilitate greater improvements in function and quality of life in PAD patients. http://dx.doi.org/10.1016/j.niox.2012.04.017 IS-8 Effect of dietary nitrate on skeletal muscle energetics in nor- moxia and hypoxia Anni Vanhatalo College of Life and Environmental Sciences, University of Exeter Hypoxia is associated with a reduction in the maximal oxidative metabolic rate of skeletal muscle which is reflected in a slowing of muscle phosphocreatine (PCr) recovery kinetics following exercise. For the same metabolic rate, there is a greater muscle metabolic perturbation (e.g., greater fall in [PCr]) during exercise performed in hypoxia compared to normoxia. Nitric oxide (NO) is a key signalling molecule for hypoxic vasodilatation and it also modulates mitochondrial O 2 consumption. Dietary nitrate intake, which increases NO bioavailability, might therefore improve O 2 delivery to active muscle and reduce O 2 demand during hypoxic exercise. It has been shown that nitrate supplementation lessens muscle metabolic perturbation during high-intensity exercise and considerably enhances exercise tolerance in moderate hypoxia. Nitrate supplementation has also been shown to abolish the reduction in the rate of PCr recovery which is typically observed in hypoxia, indicating enhanced muscle oxygenation and a restoration of muscle oxidative function. Further research is warranted to identify to what extent these effects can be attributed to changes in muscle energy metabolism and/or improved O 2 delivery. These findings indicate that dietary nitrate supplementation may have important therapeutic applications for improving skeletal muscle energetics and functional capacity in conditions where muscle O 2 delivery is compromised. http://dx.doi.org/10.1016/j.niox.2012.04.018 Session: Molecular and post-translational regula- tion of NOS enzymes IS-9 Tetrahydrobiopterin and electron transfer in NO synthase Simon Daff a , Ben Gazur a , Davide Papale a , Craig McInnes b , Raghavendar R. Morthala b , Colin L. Gibson b , Colin J. Suckling b a EASTChem School of Chemistry, University of Edinburgh, University of Strathclyde, b WestCHEM Department of Pure and Applied Chemistry, University of Strathclyde Mammalian NO synthase requires the cofactor tetrahydrobiopter- in (H 4 B) to act as an electron donor during the activation of molecular oxygen at the heme site. After donating an electron, the resultant H 4 B radical is then required to abstract an electron from the ferrous NO complex, which is generated at the end of the catalytic reaction, in order to facilitate NO release. We have recently explored the structural requirements of NO synthase for the H 4 B cofactor by studying a range of novel cofactor analogues with highly modified structures. Substitu- ents on the C6 and C7 positions of H 4 B are tolerated well, with surpris- ingly bulky pterins being able to bind and drive NO synthesis. The modified pterins have a wide range of activities and binding constants, but the main function of the cofactors in activating molecular oxygen appears to be independent of C6 and C7 modification as shown by rapid reaction studies. We have also assessed the possibility of direct electron transfer across the dimer interface between H 4 B molecules in the two NO synthase subunits. The H 4 B cofactors are within the range for facile electron transfer and present a possible mechanism for NO synthase to escape from the unreactive ferrous-NO complex, which is known to originate from product inhibition. http://dx.doi.org/10.1016/j.niox.2012.04.019 IS-10 Post-translational heme insertion into NOS and related enzymes Dennis Stuehr , Ritu Chakravarti, Arnab Ghosh, Luciana Hannibal Department of Pathobiology, Cleveland Clinic, Cleveland, OH 44195, USA Heme proteins function in biological electron transfer, substrate oxidation, and small molecule transport or detection. While heme biosynthesis is well-studied, much less is known about heme trans- port in mammalian cells, how heme becomes inserted into soluble apo-protein targets, and how these processes may be regulated. We recently found that biological amounts of nitric oxide (NO) can inhibit heme insertion into a variety of hemeproteins in mammalian cells [1]. We utilized this finding to investigate mechanisms of heme insertion and regulation, primarily using cell culture methods and initially focusing on NO synthase (NOS) as a model target. The talk will (i) present evidence that NO can act as a general regulator of cel- lular heme insertion, (ii) establish the involvement of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [2], heat shock protein-90 (hsp90) [3], and thioredoxin-1 [4] in enabling or regulating heme insertion into apo-NOS and/or other apo-hemeproteins, and (iii) dis- cuss the potential mechanisms of NO, GAPDH, hsp90, and Trx1 actions in this context. References [1] Waheed SM, Ghosh A, Chakravarti R, Biswas A, Haque MM, Panda K, Stuehr DJ. Free Rad. Biol. Med. 2010;48:1548–58. [2] Chakravarti R, Aulak KS, Fox PL, Stuehr DJ. Proc. Natl. Acad. Sci. USA 2010;107:18004–9. [3] Ghosh A, Chawla-Sarkar M, Stuehr DJ. FASEB J. 2011;25:2049–60. [4] R. Chakravarti, D.J. Stuehr, J. Biol. Chem. e-pub March 28, in press. http://dx.doi.org/10.1016/j.niox.2012.04.020 IS-11 Role of Hsp90- and Hsp70-chaperones in regulation of neuronal NO-synthase ubiquitination and proteasomal degradation Yoichi Osawa , Hwei-Ming Peng, Kelly M. Clapp, Miranda Lau, Yoshihiro Morishima Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI 48109, USA The metabolism-based inactivation of neuronal NO-synthase (nNOS) leads to the covalent alteration of the nNOS heme active site and proteasomal degradation of the inactivated nNOS. The mecha- nism of how the inactivated nNOS is selectively culled for degrada- tion is not known. We have previously shown that nNOS turnover is regulated by Hsp70/CHIP (C-terminus of Hsp70-interacting pro- tein)-dependent ubiquitination. We now show that xenobiotic-inac- tivated nNOS is selectively ubiquitinated by CHIP in an in vitro Abstracts / Nitric Oxide 27 (2012) S2–S50 S5