Russell LoBrutto

Thermodynamic and EPR studies of slowly relaxing ubisemiquinone species in the isolated bovine heart complex I

Previously, we investigated ubisemiquinone (SQ) EPR spectra associated with NADH‐ubiquinone oxidoreductase (complex I) in the tightly coupled bovine heart submitochondrial particles (SMP). Based upon their widely differing spin relaxation rate, we distinguished SQ spectra arising from three distinct SQ species, namely SQNf (fast), SQNs (slow), and SQNx (very slow). The SQNf signal was observed only in the presence of the proton electrochemical gradient ( Δ μ H + ) , while SQNs and SQNx species did not require the presence of Δ μ H + . We have now succeeded in characterizing the redox and EPR properties of SQ species in the isolated bovine heart complex I. The potentiometric redox titration of the g z,y,x = 2.00 semiquinone signal gave the redox midpoint potential (E m) at pH 7.8 for the first electron transfer step [E m1(Q/SQ)] of −45 mV and the second step [E m2(SQ/QH2)] of −63 mV. It can also be expressed as [E m(Q/QH2)] of −54 mV for the overall two electron transfer with a stability constant (K stab) of the SQ form as 2.0. These characteristics revealed the existence of a thermodynamically stable intermediate redox state, which allows this protein‐associated quinone to function as a converter between n = 1 and n = 2 electron transfer steps. The EPR spectrum of the SQ species in complex I exhibits a Gaussian‐type spectrum with the peak‐to‐peak line width of ∼6.1 G at the sample temperature of 173 K. This indicates that the SQ species is in an anionic Q ▪− state in the physiological pH range. The spin relaxation rate of the SQ species in isolated complex I is much slower than the SQ counterparts in the complex I in situ in SMP. We tentatively assigned slow relaxing anionic SQ species as SQNs, based on the monophasic power saturation profile and several fold increase of its spin relaxation rate in the presence of reduced cluster N2. The current study also suggests that the very slowly relaxing SQNx species may not be an intrinsic complex I component. The functional role of SQNs is further discussed in connection with the SQNf species defined in SMP in situ.

Modified reaction centres oxidize tyrosine in reactions that mirror photosystem II

The participation of tyrosine in the oxidation of water by photosystem II, a multisubunit enzyme complex involved in plant photosynthesis, exemplifies the significant role amino-acid side chains play in oxidation/reduction reactions in proteins. The influence of the surrounding protein on the properties of amino-acid radicals and the attributes necessary for electron transfer are not well understood. Here we report that modifications of reaction centres from the purple bacterium Rhodobacter sphaeroides result in the generation of a tyrosyl radical in a manner similar to that of photosystem II. Our design incorporates a highly oxidizing bacteriochlorophyll dimer possessing a midpoint potential of more than 0.8 V, which was achieved by altering its local environment. After substitution of tyrosine residues at positions corresponding to the tyrosyl radicals of photosystem II, optical and electron paramagnetic resonance spectra showed changes consistent with oxidation of the tyrosine. The properties of this reaction include initiation by light and coupling to proton transfer, most probably to residues near the tyrosines.

Synthesis of chlorophyll b : Localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit

BackgroundAssembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly.ResultsFluorescence of a CAO-GFP fusion protein, transiently expressed in young pea leaves, was found at the periphery of mature chloroplasts and on thylakoid membranes by confocal fluorescence microscopy. However, when membranes from partially degreened cells of Chlamydomonas reinhardtii cw15 were resolved on sucrose gradients, full-length CAO was detected by immunoblot analysis only on the chloroplast envelope inner membrane. The electron paramagnetic resonance spectrum of CAO included a resonance at g = 4.3, assigned to the predicted mononuclear iron center. Instead of a spectrum of the predicted Rieske iron-sulfur center, a nearly symmetrical, approximately 100 Gauss peak-to-trough signal was observed at g = 2.057, with a sensitivity to temperature characteristic of an iron-sulfur center. A remarkably stable radical in the protein was revealed by an isotropic, 9 Gauss peak-to-trough signal at g = 2.0042. Fragmentation of the protein after incorporation of 125I- identified a conserved tyrosine residue (Tyr-422 in Chlamydomonas and Tyr-518 in Arabidopsis) as the radical species. The radical was quenched by chlorophyll a, an indication that it may be involved in the enzymatic reaction.ConclusionCAO was found on the chloroplast envelope and thylakoid membranes in mature chloroplasts but only on the envelope inner membrane in dark-grown C. reinhardtii cells. Such localization provides further support for the envelope membranes as the initial site of Chl b synthesis and assembly of LHCs during chloroplast development. Identification of a tyrosine radical in the protein provides insight into the mechanism of Chl b synthesis.

Redox effects on the bacteriochlorophyll α-containing Fenna-Matthews-Olson protein fromChlorobium tepidum

The BChla-containing Fenna-Matthews-Olson (FMO) protein from the green sulfur bacteriumChlorobium tepidum was purified and characterized. Fluorescence spectra indicate that efficient excited state quenching occurs at neutral or oxidizing redox potentials. The major fluorescence lifetime at room temperature is approximately 60 ps in samples that are in neutral or oxidizing conditions, and approximately 2 ns in samples where the strong reductant sodium dithionite has been added. A similar change is observed in pump-probe picosecond absorbance difference experiments, where the long life time component increases after dithionite addition. A 16 Gauss wide EPR signal with g factor =2.005 is observed in samples without dithionite. This signal largely disappears upon addition of dithionite. Dithionite induces large reversibile changes in the 77 K absorbance spectra of the purified FMO protein and in whole cells. These results indicate that the FMO protein contains redox active groups, which may be involved in the regulation of energy transfer. Room temperature circular dichroism and low temperature absorption spectra show that dithionite also induces conformational or structural changes of the FMO protein complex.

Spectroscopic evidence for the presence of an iron-sulfur center similar to Fx of Photosystem I inHeliobacillus mobilis

Treatment of membranes ofHeliobacillus mobilis with high concentrations of the chaotropic agent urea resulted in the removal of the iron-sulfur centers FA and FB from the reaction center, as indicated by EPR spectra under strongly reducing conditions. In urea-treated membranes, transient absorption measurements upon a laser flash indicated a recombination between the photo-oxidized primary donor P798+ and a reduced acceptor with a time constant of 20 ms at room temperature. Benzylviologen, vitamin K-3 and methylene blue were found to accept electrons from the reduced acceptor efficiently. A differential extinction coefficient of 225–240 mM−1 cm−1 at 798 nm was determined from experiments in the presence of methylene blue. Transient absorption difference spectra between 400 and 500 nm in the presence and absence of artificial acceptors indicated that the electron acceptor involved in the 20 ms recombination has an absorption spectrum similar to that of an iron-sulfur center. This iron-sulfur center was assigned to be analogous to FX of Photosystem I. Our results provide evidence in support of the presence of FX in heliobacteria, which was proposed on the basis of the reaction center polypeptide sequence (Liebl et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7124–7128). Implications for the electron transfer pathway in the reaction center of heliobacteria are discussed.

A Cambialistic Superoxide Dismutase in the Thermophilic Photosynthetic Bacterium Chloroflexus aurantiacus

Superoxide dismutase from the thermophilic anoxygenic photosynthetic bacterium Chloroflexus aurantiacus was cloned, purified, and characterized. This protein is in the manganese- and iron-containing family of superoxide dismutases and is able to use both manganese and iron catalytically. This appears to be the only soluble superoxide dismutase in C. aurantiacus. Iron and manganese cofactors were identified by using electron paramagnetic resonance spectroscopy and were quantified by atomic absorption spectroscopy. By metal enrichment of growth media and by performing metal fidelity studies, the enzyme was found to be most efficient with manganese incorporated, yet up to 30% of the activity was retained with iron. Assimilation of iron or manganese ions into superoxide dismutase was also found to be affected by the growth conditions. This enzyme was also found to be remarkably thermostable and was resistant to H2O2 at concentrations up to 80 mM. Reactive oxygen defense mechanisms have not been previously characterized in the organisms belonging to the phylum Chloroflexi. These systems are of interest in C. aurantiacus since this bacterium lives in a hyperoxic environment and is subject to high UV radiation fluxes.

EPR Spectroscopy of VO2+-ATP Bound to Catalytic Site 3 of Chloroplast F1-ATPase from ChlamydomonasReveals Changes in Metal Ligation Resulting from Mutations to the Phosphate-binding Loop Threonine (βT168)

Site-directed mutations were made to the phosphate-binding loop threonine in the β-subunit of the chloroplast F1-ATPase in Chlamydomonas (βT168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from βT168D, βT168C, and βT168L mutants had <10% of the wild type rates, as did rates of Mg2+-ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO2+-ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51V hyperfine parameters that depend on the equatorial VO2+ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphate-binding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg2+-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg2+-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.

Probing interactions from solvent-exchangeable protons and monovalent cations with the 1,2-propanediol-1-yl radical intermediate in the reaction of dioldehydrase

The reaction of adenosylcobalamin‐dependent dioldehydrase with 1,2‐propanediol gives rise to a radical intermediate observable by EPR spectroscopy. This reaction requires a monovalent cation such as potassium ion. The radical signal arises from the formation of a radical pair comprised of the Co(II) of cob(II)alamin and a substrate‐related radical generated upon hydrogen abstraction by the 5′‐deoxyadenosyl radical. The high‐field asymmetric doublet arising from the organic radical has allowed investigation of its composition and environment through the use of EPR spectroscopic techniques. To characterize the protonation state of the oxygen substituents in the radical intermediate, X‐band EPR spectroscopy was performed in the presence of D2O and compared to the spectrum in H2O. Results indicate that the unpaired electron of the steady‐state radical couples to a proton on the C(1) hydroxyl group. Other spectroscopic experiments were performed, using either potassium or thallous ion as the activating monovalent cation, in an attempt to exploit the magnetic nature of the 205,203Tl nucleus to identify any intimate interaction of the radical intermediate with the activating cation. The radical intermediate in complex with dioldehydrase, cob(II)alamin and one of the activating monovalent cations was observed using EPR, ENDOR, and ESEEM spectroscopy. The spectroscopic evidence did not implicate a direct coordination of the activating cation and the substrate derived radical intermediate.