Substrate binding to NO-ferro-naphthalene 1,2-dioxygenase studied by high-resolution Q-band pulsed ²H-ENDOR spectroscopy

The active site of naphthalene 1,2-dioxygenase (NDO) contains a Rieske Fe-S cluster and a mononuclear non-heme iron, which are contributed by different α-subunits in the (αβ)₃ structure. The enzyme catalyzes cis-dihydroxylation of aromatic substrates, in addition to numerous other adventitious oxidation reactions. High-resolution Mims ²H-ENDOR (electron nuclear double resonance) spectra have been recorded for the NO-ferrous center of NDO bound with the substrates d₈-naphthalene, d₂-naphthalene, d₈-toluene, d₃-toluene, and d₆-benzene; samples were prepared in a D²O buffer to test for solvent-derived ligands; spectra were collected for enzymes with the Rieske diiron center in both its oxidized and reduced states. A sharp quartet ENDOR pattern from a nearby deuteron of the substrate in a major binding geometry (denoted as A) was detected for all perdeuterated substrates. Examination of the sample prepared with 1,4-di-deutero-naphthalene shows that the signal arises from D1. Analysis of two-dimensional (2-D) orientation-selective ENDOR patterns collected for this sample defined the location of the D1 deuteron, with respect to the g-frame of the iron center and the orientation of the C-D1 bond. Consideration of the orientations of naphthalene that are permitted within the constraints of these results, as supported by a novel approach to simulations of orientation-selective, 2-D ENDOR patterns for the perdeuterated naphthalene sample, which summed contributions from D1/D2/D8, disclose the geometry of the naphthalene and the Fe-NO fragment. The two deuterons of the reactive carbons, D1 and D2, are closest to the Fe atom (r_Fe-D1 ≈ 4.3 Å, r_Fe-D2 ≈ 5.0 Å), whereas D8 is farther away (r_Fe-D8 ≈ 5.3 Å). Perhaps more instructive, D1-N and D2-N distances to the O₂ surrogate, NO, are ∼2.4 and ∼3.3 Å, respectively, whereas the D8-N distance is ∼3.7 Å. The data show that benzene and the aromatic ring of toluene also sit within the substrate-binding pocket adjacent to the mononuclear Fe atom. These rings occupy a position similar to that of the "proximal" ring of naphthalene, with the closest ring deuteron being located at a distance of ∼4.3-4.4 Å from the Fe atom and with the Fe-D vector being slightly off the Fe-N(O) direction. In particular, comparison of the data for d₈-toluene and methyl-d₃-toluene shows that the methyl group of toluene points away from the Fe atom, despite observations that the oxidation of toluene occurs at the methyl group during catalysis. The Rieske cluster is reduced during both steady-state and single-turnover catalysis; therefore, the effect of its oxidation state on the geometry of substrate binding was examined. The spectra from the NDO-naphthalene complex also revealed a second binding conformation (denoted as B), in which the substrate is located ∼0.5 Å farther from the Fe atom. The relative populations of A- and B-sites are allosterically changed when the Rieske cluster is reduced. ENDOR of exchangeable protons shows that the water/hydroxide of Fe-NDO is retained upon binding NO.