The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 Å resolution or better are described of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct. The D402N enzyme has no detectable activity with neutral nitroalkanes (Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 23,[8738][8739]. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066. The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.Nitroalkane oxidase (NAO 1 ) from the soil fungus Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the corresponding aldehydes or ketones with the release of nitrite and the † This research was supported in part by grants to PFF from the NIH (GM058698) and The Welch Foundation (A-1245) and to AMO from the Offices of Biological and Environmental Research US Department of Energy, the National Center for Research Resources (2 P41 RR012408) of the NIH and from the of the US Department of Energy. Use of the National Synchrotron Light Source at Brookhaven National Laboratory was supported by the U.S. Department of Energy Office of Basic Energy Sciences, under Contract DE-AC02-98CH10886. *Corresponding authors. PFF: phone, fax, fitzpatrick@biochem.uthscsa The atomic coordinates and structure factors have been deposited with the Protein Data Bank with the corresponding file names: a) D402N NAO plus 1-nitrohexane, 3D9D; b) D402N NAO plus 1-nitrooctane, 3D9E; c) S276A NAO plus 1-nitrohexane, 3D9F; and d) wild-type NAO containing the N5-cyanohexyl FAD, 3D9G. 1 Abbreviations: NAO, nitroalkane oxidase; ACO, acyl-CoA oxidase; ACAD, acyl-CoA dehydrogenase; K ne , steady-state K m for nitroethane.

side-chain amide groups. The analysis of the C-H-O angle indicates that protein C-H donors do not show the preference for linear contacts. Although there is no tendency for linear C-H/O interactions, there are only around 3% of bifurcated C-H/O interactions. Furthermore, the analyses of the C-H/O interactions indicate an influence of simultaneous classical hydrogen bonds, especially for the tyrosine systems. The calculated electrostatic potential maps for model systems can explain the results of the crystallographic analysis. These results can be important for recognizing the C-H/O interaction of aromatic rings in the crystal structures of proteic systems.

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Aromatic–aromatic interactions have long been considered important in the self-assembly of amyloids. In spite of their importance, aromatic amino acids are not detected in every amyloid. In the present study, the occurrence and geometry of these interactions were analyzed for the amyloid structures found in the Protein Data Bank. The data confirm that aromatic amino acids are not crucial for amyloid fibril formation. In fact, aromatic–aliphatic interactions are more frequent than the aromatic–aromatic interactions. Aromatic–aliphatic interactions are present in higher numbers of structures and in certain amyloid sequences they are more frequent than aromatic–aromatic interactions. An analysis of aromatic/aromatic interactions shows different interaction geometries in intrasheet and intersheet contacts; the intrasheet aromatic–aromatic interactions are mostly parallel and displaced, while intersheet interactions are not parallel. Thus, among the aromatic–aromatic interactions there are important edge-to-face attractions in addition to parallel stacking ones.