Michael D. Clay

Nitric oxide binding at the mononuclear active site of reduced Pyrococcus furiosus superoxide reductase

Nitric oxide (NO) has been used as a substrate analog to explore the structural and electronic determinants of enzymatic superoxide reduction at the mononuclear iron active site of Pyrococcus furiosus superoxide reductase (SOR) through the use of EPR, resonance Raman, Fourier transform IR, UV-visible absorption, and variable-temperature variable-field magnetic CD spectroscopies. The NO adduct of reduced SOR is shown to have a near-axial S = 3/2 ground state with E/D = 0.06 and D = 12 ± 2 cm−1 (where D and E are the axial and rhombic zero-field splitting parameters, respectively) and the UV-visible absorption and magnetic CD spectra are dominated by an out-of-plane NO−(π*)-to-Fe3+(dπ) charge–transfer transition, polarized along the zero-field splitting axis. Resonance Raman studies indicate that the NO adduct is six-coordinate with NO ligated in a bent conformation trans to the cysteinyl S, as evidenced by the identification of ν(N–O) at 1,721 cm−1, ν(Fe–NO) at 475 cm−1, and ν(Fe–S(Cys), at 291 cm−1, via 34S and 15NO isotope shifts. The electronic and vibrational properties of the S = 3/2 {FeNO}7 unit are rationalized in terms of a limiting formulation involving a high-spin (S = 5/2) Fe3+ center antiferromagnetically coupled to a (S = 1) NO− anion, with a highly covalent Fe3+−NO− interaction. The results support a catalytic mechanism for SOR, with the first step involving oxidative addition of superoxide to form a ferric-peroxo intermediate, and indicate the important roles that the Fe spin state and the trans cysteinate ligand play in effecting superoxide reduction and peroxide release.

Heterodisulfide reductase from Methanothermobacter marburgensis contains an active-site [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction.

Heterodisulfide reductases (HDRs) from methanogenic archaea are iron–sulfur flavoproteins or hemoproteins that catalyze the reversible reduction of the heterodisulfide (CoM‐S–S‐CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM‐SH) and coenzyme B (CoB‐SH). In this work, the ground‐ and excited‐state electronic properties of the paramagnetic Fe–S clusters in Methanothermobacter marburgensis HDR have been characterized using the combination of electron paramagnetic resonance and variable‐temperature magnetic circular dichroism spectroscopies. The results confirm multiple S=1/2 [4Fe–4S]+ clusters in dithionite‐reduced HDR and reveal spectroscopically distinct S=1/2 [4Fe–4S]3+ clusters in oxidized HDR samples treated separately with the CoM‐SH and CoB‐SH cosubstrates. The active site of HDR is therefore shown to contain a [4Fe–4S] cluster that is directly involved in mediating heterodisulfide reduction. The catalytic mechanism of HDR is discussed in light of the crystallographic and spectroscopic studies of the related chloroplast ferredoxin:thioredoxin reductase class of disulfide reductases.

Spectroscopic investigation of the nickel-containing porphinoid cofactor F 430 . Comparison of the free cofactor in the +1, +2 and +3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent, red and ox forms

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F430, a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F430 in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F430, variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F430M) in the +1, +2 and +3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle π orbitals that are unique to Ni(I) forms of F430. Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d–d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F430 macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1.