Monomeric sarcosine oxidase (MSOX) is an inducible bacterial flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound FAD [8alpha-(S-cysteinyl)FAD]. This paper describes the spectroscopic and thermodynamic properties of MSOX as well as the X-ray crystallographic characterization of three new enzyme.inhibitor complexes. MSOX stabilizes the anionic form of the oxidized flavin (pK(a) = 8.3 versus 10.4 with free FAD), forms a thermodynamically stable flavin radical, and stabilizes the anionic form of the radical (pK(a) < 6 versus pK(a) = 8.3 with free FAD). MSOX forms a covalent flavin.sulfite complex, but there appears to be a significant kinetic barrier against complex formation. Active site binding determinants were probed in thermodynamic studies with various substrate analogues whose binding was found to perturb the flavin absorption spectrum and inhibit MSOX activity. The carboxyl group of sarcosine is essential for binding since none is observed with simple amines. The amino group of sarcosine is not essential, but binding affinity depends on the nature of the substitution (CH(3)XCH(2)CO(2)(-), X = CH(2) < O < S < Se < Te), an effect which has been attributed to differences in the strength of donor-pi interactions. MSOX probably binds the zwitterionic form of sarcosine, as judged by the spectrally similar complexes formed with dimethylthioacetate [(CH(3))(2)S(+)CH(2)CO(2)(-)] and dimethylglycine (K(d) = 20.5 and 17.4 mM, respectively) and by the crystal structure of the latter. The methyl group of sarcosine is not essential but does contribute to binding affinity. The methyl group contribution varied from -3.79 to -0.65 kcal/mol with CH(3)XCH(2)CO(2)(-) depending on the nature of the heteroatom (NH(2)(+) > O > S) and appeared to be inversely correlated with heteroatom electron density. Charge-transfer complexes are formed with MSOX and CH(3)XCH(2)CO(2)(-) when X = S, Se, or Te. An excellent linear correlation is observed between the energy of the charge transfer bands and the one-electron reduction potentials of the ligands. The presence of a sulfur, selenium, or telurium atom identically positioned with respect to the flavin ring is confirmed by X-ray crystallography, although the increased atomic radius of S < Se < Te appears to simultaneously favor an alternate binding position for the heavier atoms. Although L-proline is a poor substrate, aromatic heterocyclic carboxylates containing a five-membered ring and various heteroatoms (X = NH, O, S) are good ligands (K(d, X=NH) = 1.37 mM) and form charge-transfer complexes with MSOX. The energy of the charge-transfer bands (S > O >> NH) is linearly correlated with the one-electron ionization potentials of the corresponding heterocyclic rings.

The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ), and the subsequent oxidative deamination of substrate amines. Because the same active-site pocket must facilitate both reactions, individual active-site residues may serve multiple roles. We have examined the roles of a strictly-conserved active-site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically, and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild-type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxyintermediates in cofactor biogenesis, but also indicates the critical control of oxidation chemistry that can be conferred by a single active-site residue.Copper amine oxidases (CAO) comprise a family of enzymes that are found almost ubiquitously among aerobic prokaryotic and eukaryotic organisms. These enzymes catalyze the oxidation of primary amines to aldehydes and reduction of dioxygen to hydrogen peroxide via a ping-pong mechanism involving a covalently-bound redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ) and a copper ion, Cu(II) (1,2). In conjunction with the multiple catalytic turnovers catalyzed by the mature CAOs, these enzymes catalyze a second, single turnover reaction. The latter process involves the reaction of an active-site tyrosine and molecular oxygen, in a copper-dependent manner, to generate the TPQ cofactor, Scheme 1 (3-5). The active-site Tyr that leads to TPQ (position 405 in the CAO from Hansenula polymorpha) is absolutely conserved among CAO family members and is contained within the consensus sequence Thr-X-X-Asn-Tyr-Asp/Glu (4). Other strictlyconserved residues include three histidine residues (His456, His458 and His624) that form † This work was supported by grants from the National Institutes of Health: GM31611 to FSM, GM039296 to JPK, and GM63414-01 to J.L.D. Use of the Advanced Photon Source supported by the U.S. DOE, Office of Basic Energy Sciences, Contract No. W-31-109-ENG-38.¶ To whom correspondence should be addressed: JPK: Phone: (510) 642-2668; Fax: (510) Supporting Information This includes occupancies and temperature factors of the final model (Table S1), and interactions and associated water molecules in the final model (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 August 31. There are a number of possible roles that can be considered for Y305 in the course of both catalytic turnover and cofactor biogenesis. For amine oxidation, the close proximity of Tyr 305 to the O-4 of the TPQ anion ( Figure 1A) suggests a short, strong hydrogen bond that may be expected to play a role in charge localization and stabilization. The latter are expected to facilitate the formation of a...