The crystal structure of dimeric Fe(III) superoxide dismutase (SOD) from Escherichia coli (3006 protein atoms, 2 irons, and 281 solvents) has been refined to an R of 0.184 using all observed data between 40.0 and 1.85 A (34,879 reflections). Features of this structure are compared with the refined structure of MnSOD from Thermus thermophilus. The coordination geometry at the Fe site is distorted trigonal bipyramidal, with axial ligands His26 and solvent (proposed to be OH-), and in-plane ligands His73, Asp156, and His160. Reduction of crystals to the Fe(II) state does not result in significant changes in metal-ligand geometry (R = 0.188 for data between 40.0 and 1.80 A). The arrangement of iron ligands in Fe(II) and Fe(III)SOD closely matches the Mn coordination found in MnSOD from T. thermophilus [Ludwig, M.L., Metzger, A.L., Pattridge, K.A., & Stallings, W.C. (1991) J. Mol. Biol. 219, 335-358]. Structures of the Fe(III) azide (40.0-1.8 A, R = 0.186) and Mn(III) azide (20.0-1.8 A, R = 0.179) complexes, reported here, reveal azide bound as a sixth ligand with distorted octahedral geometry at the metal; the in-plane ligand-Fe-ligand and ligand-Mn-ligand angles change by 20-30 degrees to coordinate azide as a sixth ligand. However, the positions of the distal azide nitrogens are different in the FeSOD and MnSOD complexes. The geometries of the Fe(III), Fe(II), and Fe(III)-azide species suggest a reaction mechanism for superoxide dismutation in which the metal alternates between five- and six-coordination. A reaction scheme in which the ligated solvent acts as a proton acceptor in the first half-reaction [formation of Fe(II) and oxygen] is consistent with the pH dependence of the kinetic parameters and spectroscopic properties of Fe superoxide dismutase.

The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO) superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H+ and e- transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba3-type cytochrome c oxidase from Thermus thermophilus at 1.8 Å resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O2-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the CuB atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fea3 and CuB atoms that is best modeled as peroxide. The structure of ba3-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba3-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.

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The catalysis of superoxide dismutation ( 2 0 1 + 2H+ -H202 + 0,) by manganese superoxide dismutase (MnSOD) from Thermus Thermophilus was examined by stopped-flow spectrophotometry. As found earlier by McAdam et al. [McAdam, M. E.; Fox, R. A.; Lavelle, F.; Fielden, E. M. Biochem. J . 1977, 165, 81-87], decay curves of 01 in the presence of MnSOD from Bacillus Stearothermophilus are characterized by three distinct phases: rapid disappearance of 02-(the "burst" phase), a period of approximately zero-order disappearance of 02-(the "steady-state" phase), and a very rapid depletion of 02-toward the end of the reaction. The enzyme from T. Thermophilus shows a similar kinetic pattern, and our data provide a chemical explanation for this behavior: The molar consumption of 0; in the burst phase is ([O2-Ie/[MnlT) -80. The magnitude of the burst is decreased -2.5-fold in D20, whereas the zero-order phase is the same in both solvents. This indicates that proton transfer is probably the rate-limiting step when the enzyme is saturated with 0; and that the reaction by which inactive enzyme returns to active enzyme is not limited by proton transfer. At low temperatures (2-6 "C) in D20, the overall reaction was sufficiently slow to allow observation of spectral changes associated with the metal chromophore during the steady state, and we were able to obtain an absorption spectrum of the enzyme during this period. This was assigned to the inactive form of the enzyme and is characterized by a band near 650 nm (c -230 [Mnl-' cm-I) and a band near 410 nm (c -700 [Mnl-' cm-I). We speculate that inactivation of the enzyme occurs by oxidative addition of 0; to Mn(II), within a Michaelis complex, forming a cyclic peroxo complex of Mn(II1) with the reverse of this reaction yielding active enzyme. k j -650 S-' Mn":02-+ Mn"':o22k-5 -10 s-' A reaction scheme composed of a cyclic redox process, as described previously for the FeSOD of Escherichia coli [Bull, C . ; Fee, J. A. J . Am. Chem. SOC. 1985, 107, 3295-33041, and the above reversible side reaction adequately account for the kinetic behavior of MnSODs.In previous communications we described the steady-state kinetic properties of the iron-containing superoxide dismutase from E. coli,' of Cu/Zn-containing superoxide dismutase (SOD35) from bovine tissues,* and of FeEDTA.3v35 In this paper we describe our studies with the manganese-containing protein from Thermus thermophilus.There are three types of superoxide dismutases as determined by the metal involved in the catalysis of reaction 1: Cu, Fe, and Mn. The general properties, distribution, and possible biological (1) function of these proteins have been discussed in a large number of review and discussion papers (cf. Ref 1 of ref 1 for reviews). The present work is concerned with manganese-containing superoxide dismutases, and the object of study is the protein from Thermus thermophilus. This protein is a tetramer of 21 kDa subunits, each of which binds, on the average, -0.6 Mn(II1) ions. Solutions of the protein have a reddish-pur...

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier Mössbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.