James A. Fee

Evidence that superoxide dismutase plays a role in protecting red blood cells against peroxidative hemolysis.

Abstract We have carried out kinetic studies of the dialuric acid induced hemolysis of vitamin E deficient rat erythrocytes, and have shown that with a careful choice of conditions and in the presence of catalase a very substantial protective effect of externally added superoxide dismutase can be observed.

Nuclear magnetic relaxation dispersion in protein solutions: V. Bovine erythrocyte superoxide dismutase☆☆☆

Abstract The paramagnetic contribution Rp to the spin-lattice relaxation rate of solvent protons in aqueous solutions of bovine erythrocyte superoxide dismutase has been measured for magnetic fields in the range 5 Oe to 12 kOe and temperatures in the range 0 °C to 25 °C. We find a peak in Rp as a function of magnetic field, as has been observed recently by several investigators for Mn2+-macromolecular complexes, indicating that the spin-lattice relaxation time τs of the Cu2+ electron spin moments is magnetic field dependent. τs is approx. 5·10−10 s at low field and begins to increase above approx. 750 Oe. From the magnitude of Rp and its qualitative behavior with magnetic field and temperature, combined with previous EPR and inhibition studies, we conclude that there is at least one rapidly exchanging water molecule (exchange time τM in the range 4·10−6 to 10−8 s) on each of the two Cu2+ per protein molecule. We speculate that the water binding sites on the Cu2+, with a Cu—O bond distance approx. 2.0 A, are similarly accessible to the superoxide anion and are the loci for the catalytic activity of the enzyme.

The iron electron-nuclear double resonance (ENDOR) of two-iron ferredoxins from spinach, parsley, pig adrenal cortex and Pseudomonas putida ☆

Abstract The iron electron-nuclear double resonance (ENDOR) spectra of reduced iron-sulfur proteins (two-iron ferredoxins) from spinach, parsley, pig adrenal cortex and Pseudomonas putida unequivocally show two inequivalent iron atoms at the active sites of each of these proteins. The frequencies of the ENDOR lines establish the total electronic spin in the ground state to be S = 1 2 . The principal values of the hyperfine tensor have been determined for each of the iron atoms and these values are consistent with and lend considerable support to the model of a high-spin Fe(III) atom and a high-spin Fe(II) atom antiferromagnetically coupled to form an S = 1 2 system. The measured principal axis components of the effective hyperfine tensors for S = 1 2 are as follows (1 and 2 refer to the inequivalent iron sites): Site 1 ( Fe ( III )) Site 2 ( Fe ( II )) A x A y ′ A z ′ A x A y A z Spinach 51 ± 1 50 − 7 +2 42 ± 1.5 ? ? 35.5 ± 2 MHz Parsley 51 ± 1 50 − 7 +2 42 ± 2 ? ? 34.5 ± 2.5 MHz A ⊥ A ⊥ ′ A z A ⊥ A ⊥ ′ A z Adrenodoxin 50 ± 1 56 − 3 +1 43 +2 − 1 17 ± 4 24 ∓ 4 35 ± 1.5 MHz Putidaredoxin 50 ± 1.5 56 − 3 +1 43 +2 − 1 17 ± 4 24 ∓ 4 35 ± 1.5 MHz These data are consistent with Site 1 being ferric and Site 2, ferrous iron. The primes indicate that the A -tensor principal axes for Site 1 (Fe(III)) are apparently rotated about the x -axis with respect to the g -tensor axes by an angle θ (20° ⩽ θ ⩽ 40°). The orientations of the A -tensors for Site 2 (Fe(II)) have not been determined and hence the values presented are the observed values of the A -tensors along the x , y , and z -axes of the g -tensor for this complex. A brief introduction to the theory of ENDOR is given.

Stopped flow spectrophotometric observation of superoxide dismutation in aqueous solution.

There are few ways to develop a high concentration of superoxide in solutions of physiological significance, and by far the most useful of these, pulsed radiolysis of oxygenated solutions [1 ], requires very specialized and expensive research equipment. One of the long term goals of this laboratory has been to develop alternative procedures for examining the chemistry of superoxide ion in aqueous solution [2]. We now report some preliminary results obtained with a stopped-flow spectrophotometric system which mixes superoxide, stabilized in aprotic solvents, into aqueous solutions and allows direct photometric observation of the superoxide. In this preliminary communication we present results on (a) the second-order dismutation of superoxide in the pH 7-11 range, (b) the kinetic behavior of bovine superoxide dismutase in this pH region, and (c) the decay of O~ in the presence of H202 which show that the Haber-Weiss [3] reaction

The cytochrome ba3 oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping

The heme-copper oxygen reductases are redox-driven proton pumps that generate a proton motive force in both prokaryotes and mitochondria. These enzymes have been divided into 3 evolutionarily related groups: the A-, B- and C-families. Most experimental work on proton-pumping mechanisms has been performed with members of the A-family. These enzymes require 2 proton input pathways (D- and K-channels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the D-channel transporting all pumped protons. In this work we use site-directed mutagenesis to demonstrate that the ba3 oxygen reductase from Thermus thermophilus, a representative of the B-family, does not contain a D-channel. Rather, it utilizes only 1 proton input channel, analogous to that of the A-family K-channel, and it delivers protons to the active site for both O2 chemistry and proton pumping. Comparison of available subunit I sequences reveals that the only structural elements conserved within the oxygen reductase families that could perform these functions are active-site components, namely the covalently linked histidine-tyrosine, the CuB and its ligands, and the active-site heme and its ligands. Therefore, our data suggest that all oxygen reductases perform the same chemical reactions for oxygen reduction and comprise the essential elements of the proton-pumping mechanism (e.g., the proton-loading and kinetic-gating sites). These sites, however, cannot be located within the D-channel. These results along with structural considerations point to the A-propionate region of the active-site heme and surrounding water molecules as the proton-loading site.

The CuA domain of Thermus thermophilus ba3-type cytochrome c oxidase at 1.6 A resolution.

The structure of the CuA-containing, extracellular domain of Thermus thermophilus ba3-type cytochrome c oxidase has been determined to 1.6 Å resolution using multiple X-ray wavelength anomalous dispersion (MAD). The Cu2S2 cluster forms a planar rhombus with a copper–copper distance of 2.51 ± 0.03 Å. X-ray absorption fine-structure (EXAFS) studies show that this distance is unchanged by crystallization. The CuA center is asymmetrical; one copper is tetrahedrally coordinated to two bridging cysteine thiolates, one histidine nitrogen and one methionine sulfur, while the other is trigonally coordinated by the two cysteine thiolates and a histidine nitrogen. Combined sequence–structure alignment of amino acid sequences reveals conserved interactions between cytochrome c oxidase subunits I and II.

The magnetic susceptibility of spinach ferredoxin from 77–250°K: A measurement of the antiferromagnetic coupling between the two iron atoms☆

Abstract The magnetic susceptibility of oxidized and reduced spinach ferredoxin has been measured over the temperature range 77–250°K. Anomalous behavior is observed in both oxidation states and the data can be interpreted by assuming an exchange interaction between the metal ions. The exchange constant is estimated to be 183 cm −1 in oxidized ferredoxin and ≤ 100 cm −1 in reduced ferredoxin.

High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment.

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.

Regulation of sod genes in Escherichia coli : relevance to superoxide dismutase function

This review is concerned with the effects of environmental perturbations on the expression of the two superoxide dismutase (SOD) genes in Escherichia coli (sodA, MnSOD; sodB, FeSOD). Early studies using SOD activity, showed that MnSOD levels respond to changes in oxygen tension, type of substrate, redox active compounds, iron concentration, the nature of the terminal oxidant, and the redox potential of the medium. FeSOD levels appeared nominally insensitive to these perturbations. More recent molecular genetic studies revealed that sodA expression is subject to regulation by three major regulatory systems: fur (ferric uptake regulation) and arcA arcB (aerobic respiratory control) mediate repression of sodA, while a relatively new system, soxR soxS (superoxide response), mediates activation of sodA expression. By contrast, sodB expression, which is much less studied at this time, appears to be positively activated in trans by fur. A rudimentary gene regulation model is presented which rationalizes past observations, is experimentally testable, and should serve as a guide to future research in this area.

Structure-Function Relationships in Iron and Manganese Superoxide Dismutases

Using the complete sequences for MnSOD from Thermus thermophilus and for FeSOD from E. coli, structural models for both oxidized enzymes have been refined, the Mn protein to an R of 0.186 for all data between 10.0 and 1.8 A, and the Fe protein to an R of 0.22 for data between 10.0 and 2.5 A. The results of the refinements support the presence of a solvent as a fifth ligand to Mn(III) and Fe(III) and a coordination geometry that is close to trigonal bipyramidal. The putative substrate-entry channel is comprised of residues from both subunits of the dimer; several basic residues that are conserved may facilitate approach of O2-, while other conserved residues maintain interchain packing interactions. Analysis of the azide complex of Fe(III) dismutase suggests that during turnover O2- binds to the metal at a sixth coordination site without displacing the solvent ligand. Because crystals reduced with dithionite show no evidence for displacement of the protein ligands, the redox-linked proton acceptor (C. Bull and J.A. Fee (1985), Journal of the American Chemistry Society 107, 3295-3304) is unlikely to be one of the histidines which bind the metal ion. Structural, kinetic, titration, and spectroscopic data can be accommodated in a mechanistic scheme which accounts for the differential titration behaviour of the Fe(III) and Fe(II) enzymes at neutral and high pH.