Geometries and Electronic Structures of Cyanide Adducts of the Non-Heme Iron Active Site of Superoxide Reductases:  Vibrational and ENDOR Studies

Abstract

We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide, and have used vibrational and ENDOR spectroscopies to study the properties of the active-site paramagnetic iron center. Addition of cyanide changes the active-site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13 CN, C 15 N, and 13 C 15 N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle ∼175°) and two distinct bent conformations (Fe-C-N angles < 140°). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13 C-ENDOR spectra for the 13 CN-bound ferric active sites in both 1Fe-and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13 C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123° (C3), 133°( C2), taking a reference value from vibrational studies of 175° (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.

Superoxide Dismutases and Superoxide Reductases

“…Thus far, only a few SORs were studied with some details: six are from Bacteria, the 2Fe-SORs from anaerobic sulfate reducing bacteria ( D. vulgaris , 367b , 372c , 384 D. baarsii , 370b , 372d , 372g , 385 and D. desulfuricans ATCC 27774 363 , 368b ), the 1Fe-SORs from the microaerophilic bacteria Treponema pallidum ( 372a , 372i , 385b , 386 ) and denticola , 387 and the sulfate-reducing bacterium D. gigas . 362 , 367a Within Archaea, they are from the hyperthermophiles (1Fe and 2Fe-SORs from A. fulgidus , 368c , 372b , 372e , 372f , 388 1Fe-SORs from Pyrococcus furiosus , 370a , 389 Ignicoccus hospitalis , 383 and Nanoarchaeum equitans ( 372h )).…”

Understanding the Mechanism of Superoxide Reductase Promoted Reduction of Superoxide

“…[32,33] Consistent with an inner sphere pathway, exogenous ligands such as azide, nitric oxide, and cyanide have also been shown to bind to the iron site of SOR. [34][35][36] The putative peroxide intermediate displays a chargetransfer band at 600 (~3500  -1 cm -1 ) nm and releases H 2 O 2 at a rate of 40-50 s -1 . [32,33,37] The intense low-energy charge-transfer (CT) band was originally assigned as a combination peroxide/sulfur-to-metal charge-transfer transition, [32] but more recently it was suggested that this band is too low in energy for the peroxide to be involved.…”

Sulfur K-Edge X-ray Absorption Spectroscopy and Density Functional Theory Calculations on Superoxide Reductase: Role of the Axial Thiolate in Reactivity

“…The decay of this intermediate involved a diffusion controlled protonation from its pH dependence and a deuterium isotope effect of 2. Lys 14 has been identified as a participating 2 nd sphere residue (mutation of this residue decreases the turnover rate by a factor of [20][21][22][23][24][25][26][27][28][29][30] and has been proposed to assist superoxide binding to the active site. 19,20 Though no intermediate has been trapped with the physiological substrate superoxide, Olivier et.…”