C are repaired by the AfAlkA base excision repair glycosylase of Archaeoglobus fulgidus, suggesting a different repair mechanism for these lesions in the third domain of life. In addition, AfAlkA was found to effect a robust excision of 1,N 6 -ethenoadenine. We present a high-resolution crystal structure of AfAlkA, which, together with the characterization of several sitedirected mutants, forms a molecular rationalization for the newly discovered base excision activity.

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Nitric oxide ( NO ) produced by mammalian nitric oxide synthases ( mNOS s) is an important mediator in a variety of physiological functions. Crystal structures of mNOS s have shown strong conservation of the active‐site residue Val567 (numbering for rat neuronal NOS , nNOS ). NOS ‐like proteins have been identified in several bacterial pathogens, and these display striking sequence identity to the oxygenase domain of mNOS ( NOS oxy), with the exception of a Val to Ile mutation at the active site. Preliminary studies have highlighted the importance of this Val residue in NO ‐binding, substrate recognition, and oxidation in mNOS s. To further elucidate the role of this valine in substrate and substrate analogue recognition, we generated five Val567 mutants of the oxygenase domain of the neuronal NOS ( nNOS oxy) and used UV‐visible and EPR spectroscopy to investigate the effects of these mutations on the heme distal environment, the stability of the heme‐Fe II ‐ CO complexes, and the binding of a series of substrate analogues. Our results are consistent with Val567 playing an important role in preserving the integrity of the active site for substrate binding, stability of heme‐bound gaseous ligands, and potential NO production.