Tewfik Soulimane

Structure and mechanism of the aberrant ba(3)-cytochrome c oxidase from thermus thermophilus.

Cytochrome c oxidase is a respiratory enzyme catalysing the energy‐conserving reduction of molecular oxygen to water. The crystal structure of the ba3‐cytochrome c oxidase from Thermus thermophilus has been determined to 2.4 Å resolution using multiple anomalous dispersion (MAD) phasing and led to the discovery of a novel subunit IIa. A structure‐based sequence alignment of this phylogenetically very distant oxidase with the other structurally known cytochrome oxidases leads to the identification of sequence motifs and residues that seem to be indispensable for the function of the haem copper oxidases, e.g. a new electron transfer pathway leading directly from CuA to CuB. Specific features of the ba3‐oxidase include an extended oxygen input channel, which leads directly to the active site, the presence of only one oxygen atom (O2−, OH− or H2O) as bridging ligand at the active site and the mainly hydrophobic character of the interactions that stabilize the electron transfer complex between this oxidase and its substrate cytochrome c. New aspects of the proton pumping mechanism could be identified.

On the Mechanism of Inhibition of Cytochrome c Oxidase by Nitric Oxide

The mechanism of inhibition of cytochrome (cyt) c oxidase by nitric oxide (NO) has been investigated by stopped flow transient spectroscopy and singular value decomposition analysis. Following the time course of cyt c oxidation at different O2/NO ratios, we observed that the onset of inhibition: (i) is fast and at a high NO concentration is complete during the first turnover; (ii) is sensitive to the O2/NO ratio; and (iii) is independent of incubation time of the oxidized enzyme with NO. Analysis of the reaction kinetics and computer simulations support the conclusion that inhibition occurs via binding of NO to a turnover intermediate with a partially reduced cyt a3-CuB binuclear center. The inhibited enzyme has the optical spectrum typical of NO bound to reduced cyt a3. Reversal of inhibition in the presence of O2 does not involve a direct reaction of O2 with NO while bound at the binuclear center, since recovery of activity occurs at the rate of NO dissociation (k = 0.13 s−1), as determined in the absence of O2 using hemoglobin as a NO scavenger. We propose that removal of NO from the medium is associated with reactivation of the enzyme via a relatively fast thermal dissociation of NO from the reduced cyt a3-CuB center.

A novel free-mounting system for protein crystals: transformation and improvement of diffraction power by accurately controlled humidity changes

A novel device for capillary-free mounting of protein crystals is described. A controlled stream of air allows an accurate adjustment of the humidity at the crystal. The crystal is held on the tip of a micropipette. With a video system (CCD camera), the two-dimensional shadow projections of crystals can be recorded for optical analysis. Instead of the micropipette, a standard loop can also be used. Experiments and results for different crystal systems demonstrate the use of this method, also in combination with shock-freezing, to improve crystal order. Working with oxygen-free gases offers the possibility of crystal measurements under anaerobic conditions. Furthermore, the controlled application of arbitrary volatile substances with the gas stream is practicable.

Time-resolved generation of a membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus Evidence for reduction-induced opening of the binuclear center

ba 3‐type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash‐induced generation of a membrane potential by the enzyme has been studied in a μs/ms time scale with Ru(II)‐tris‐bipyridyl complex (RuBpy) as a photoreductant. Flash‐induced single electron reduction of the aerobically oxidized ba 3 by RuBpy results in two phases of membrane potential generation by the enzyme with τ values of about 20 and 300 μs at pH 8 and 23°C. Spectrophotometric experiments show that oxidized ba 3 reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide. At the same time, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H2O2 with a second order rate constant of ∼2×103 M−1 s−1. The data indicate that single electron reduction of ba 3 oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba 3 is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba 3 oxidase as compared to the oxidases of the aa 3‐type.ba3-type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash-induced generation of a membrane potential by the enzyme has been studied in a microsecond/ms time scale with Ru(II)-tris-bipyridyl complex (RuBpy) as a photoreductant. Flash-induced single electron reduction of the aerobically oxidized ba3 by RuBpy results in two phases of membrane potential generation by the enzyme with tau values of about 20 and 300 microseconds at pH 8 and 23 degrees C. Spectrophotometric experiments show that oxidized ba3 reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide. At the same time, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H2O2 with a second order rate constant of approximately 2 x 10(3) M-1 s-1. The data indicate that single electron reduction of ba3 oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba3 is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba3 oxidase as compared to the oxidases of the aa3-type.

Three-dimensional crystals of cytochrome-c oxidase from Thermus thermophilus diffracting to 3.8 Å resolution

The ba 3‐type cytochrome‐c oxidase from Thermus thermophilus has been crystallized in its native form. Crystallization was achieved by the batch and the vapour diffusion sitting drop methods using polyethylene glycol monomethyl ether 2000 as precipitating agent in the presence of octyl‐β‐d‐thioglucoside as detergent. The crystals diffract to 3.8 Å, belong to the space group P2 or P21 and have unit cell dimensions of ; ; and β = 104.4°. The asymmetric unit contains two ba 3‐type oxidase molecules.

Resonance Raman spectroscopic study of the caa3 oxidase from Thermus thermophilus

The terminal caa3 oxidase of Thermus thermophilus has been studied by resonance Raman spectroscopy. Using different excitation wavelengths in the Soret band region, it was possible to disentangle the resonance Raman spectra of the fully oxidized and fully reduced state in terms of the component spectra of the individual hemes a, a3, and c. For the heme a and a3 groups, the spectra reveal only minor differences compared to those of beef heart cytochrome c oxidase attributable to subtle modifications of the heme environment. These differences are not more pronounced than those between the oxidases from beef heart and Paracoccus denitrificans confirming the view that this oxidase of Th. thermophilus is a typical member of the aa3 oxidase superfamily. The heme c component spectra display far-reaching similarities with those of c-type cytochromes which serve as mobile electron carriers in the respiratory chain. These results imply that caa3 oxidase represents an integrated version of the noncovalent redox complex between cytochrome c and cytochrome c oxidase in higher organisms. On the other hand, the structural changes of cytochrome c in the noncovalent complex have no counterpart in the heme c component of the caa3 oxidase indicating a specific cytochrome c binding site for the mitochondrial enzyme.

The active site structure of ba3 oxidase from Thermus thermophilus studied by resonance Raman spectroscopy

The ba3 cytochrome oxidase from Thermus thermophilus was studied by resonance Raman spectroscopy. The component spectra of both heme groups were determined by using different excitation wavelengths. In the ferric state the heme a3 group reveals resonance Raman marker bands characteristic for two high spin species with the heme iron in an in-plane and an out-of-plane configuration that reflects a coordination equilibrium. This equilibrium obviously results from protonation of one of the axial ligands that is ascribed to a hydroxide. Coordination by its protonated form, a water molecule, may be too weak to keep the heme iron in the porphyrin plane. The corresponding Fe-OH2 stretching mode was attributed to a weak H/D-sensitive band at 464 cm(-1). The coordination equilibrium not only depends on the pH but is also affected by the buffer, the salt concentration, and the binding of the natural redox partner cytochrome c552. These changes of the coordination equilibrium are attributed to the perturbation of the hydrogen bonding network at the catalytic center that is connected to the protein surface via a relay of hydrogen bonds. Environmental changes at the catalytic site are sensitively reflected by the formyl stretching of heme a3. The unique structural properties of the ba3 oxidase may be related to the unusual proton pump efficiency and heme a3 redox potential.

Ba3-Type cytochrome c oxidase from Thermus thermophilus: Purification, crystallization and crystal transformation

Cytochrome c oxidase, the terminal complex of the respiratory chain of mitochondria and many bacteria, catalyzes the electron transfer from cytochrome c to molecular oxygen, thereby reducing the latter to water. Thermus thermophilus HB8 (ATCC 27634) is an extremely thermophilic gram negative bacterium. It grows at temperatures between 47° and 85°C. Depending on the fermentation conditions and the 0 2 -supply, this organism expresses two different oxidases known as the caa 3 -and the ba 3 -type. This chapter describes a new procedure for the preparation of monodispersed highly active ba 3 -type cytochrome c oxidase from Thermus thermophilus. The enzyme is isolated from the membrane using Triton X-100 and purified by subsequent anion-exchange chromatographies first in Triton X-100 and then in dodecyl-β-D-maltoside. The best crystals were grown in nonyl-β-D-glucoside using PEG 2000 as precipitant. However, these crystals show drastic changes in their cell constants if measured in a standard capillary and a relatively weak diffraction power in their native (as grown) state. In order to obtain a useful dataset, it was necessary to transform these crystals by dehydration.

Transient Spectroscopy of the Reaction between Cytochrome c Oxidase and Nitric Oxide

It is of great interest, though somewhat puzzling, that nitric oxide (NO) can play simultaneously the dual role of messenger (Moncada et al., 1991), and inhibitor (Bredt and Snider, 1994; Nathan, 1992). The balance between these two functions will depend on a number of parameters, ranging from the cell-tissues pathophysiological state to the relative concentration in vivo of O2 and NO.