An unconventional copper protein required for cytochrome c oxidase respiratory function under extreme acidic conditions.

Very little is known about the processes used by acidophile organisms to preserve stability and function of respiratory pathways. Here, we reveal a potential strategy of these organisms for protecting and keeping functional key enzymes under extreme conditions. Using Acidithiobacillus ferrooxidans, we have identified a protein belonging to a new cupredoxin subfamily, AcoP, for "acidophile CcO partner," which is required for the cytochrome c oxidase (CcO) function. We show that it is a multifunctional copper protein with at least two roles as follows: (i) as a chaperone-like protein involved in the protection of the Cu(A) center of the CcO complex and (ii) as a linker between the periplasmic cytochrome c and the inner membrane cytochrome c oxidase. It could represent an interesting model for investigating the multifunctionality of proteins known to be crucial in pathways of energy metabolism.

Characterization of a new periplasmic single-domain rhodanese encoded by a sulfur-regulated gene in a hyperthermophilic bacterium Aquifex aeolicus.

Rhodaneses (thiosulfate cyanide sulfurtransferases) are enzymes involved in the production of the sulfur in sulfane form, which has been suggested to be the relevant biologically active sulfur species. Rhodanese domains occur in the three major domains of life. We have characterized a new periplasmic single-domain rhodanese from a hyperthermophile bacterium, Aquifex aeolicus, with thiosulfate:cyanide transferase activity, Aq-1599. The oligomeric organization of the enzyme is stabilized by a disulfide bridge. To date this is the first characterization from a hyperthermophilic bacterium of a periplasmic sulfurtransferase with a disulfide bridge. The aq-1599 gene belongs to an operon that also contains a gene for a prepilin peptidase and that is up-regulated when sulfur is used as electron acceptor. Finally, we have observed a sulfur-dependent bacterial adherence linked to an absence of flagellin suggesting a possible role for sulfur detection by A. aeolicus.

A new iron-oxidizing/O2-reducing supercomplex spanning both inner and outer membranes, isolated from the extreme acidophile Acidithiobacillus ferrooxidans.

The iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans involves various metalloenzymes. Here we demonstrate that the oxygen reduction pathway from ferrous iron (named downhill pathway) is organized as a supercomplex constituted of proteins located in the outer and inner membranes as well as in the periplasm. For the first time, the outer membrane-bound cytochrome c Cyc2 was purified, and we showed that it is responsible for iron oxidation and determined that its redox potential is the highest measured to date for a cytochrome c. The organization of metalloproteins inside the supramolecular structure was specified by protein-protein interaction experiments. The isolated complex spanning the two membranes had iron oxidase as well as oxygen reductase activities, indicating functional electron transfer between the first iron electron acceptor, Cyc2, and the Cu(A) center of cytochrome c oxidase aa(3). This is the first characterization of a respirasome from an acidophilic bacterium. In Acidithiobacillus ferrooxidans,O(2) reduction from ferrous iron must be coupled to the energy-consuming reduction of NAD(+)(P) from ferrous iron (uphill pathway) required for CO(2) fixation and other anabolic processes. Besides the proteins involved in the O(2) reduction, there were additional proteins in the supercomplex, involved in uphill pathway (bc complex and cytochrome Cyc(42)), suggesting a possible physical link between these two pathways.

First characterisation of the active oligomer form of sulfur oxygenase reductase from the bacterium Aquifex aeolicus.

Sulfur oxygenase reductase (SOR) enzyme is responsible for the initial oxidation step of elemental sulfur in archaea. Curiously, Aquifex aeolicus, a hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium, has the SOR-encoding gene in its genome. We showed, for the first time the presence of the SOR enzyme in A. aeolicus, its gene was cloned and recombinantly expressed in Escherichia coli and the protein was purified and characterised. It is a 16 homo-oligomer of approximately 600 kDa that contains iron atoms indispensable for the enzyme activity. The optimal temperature of SOR activity is 80 degrees C and it is inactive at 20 degrees C. Studies of the factors involved in getting the fully active molecule at high temperature show clearly that (1) incubation at high temperature induces more homogeneous form of the enzyme, (2) conformational changes observed at high temperature are required to get the fully active molecule and (3) acquisition of an active conformation induced by the temperature seems to be more important than the subunit number. Differences between A. aeolicus SOR and the archaea SORs are described.

A new sulfurtransferase from the hyperthermophilic bacterium Aquifex aeolicus. Being single is not so simple when temperature gets high.

Sulfur is a functionally important element of living matter. Rhodanese is involved in the enzymatic production of the sulfane sulfur which has been suggested as the biological relevant active sulfur species. Rhodanese domains are ubiquitous structural modules occurring in the three major evolutionary phyla. We characterized a new single-domain rhodanese with a thiosulfate : cyanide transferase activity, Aq-477. Aq-477 can also use tetrathionate and polysulfide. Thermoactivity and thermostability studies show that in solution Aquifex sulfurtranferase exists in equilibrium between monomers, dimers and tetramers, shifting to the tetrameric state in the presence of substrate. We show that oligomerization is important for thermostability and thermoactivity. This is the first characterization of a sulfurtransferase from a hyperthermophilic bacterium, which moreover presents a tetrameric organization. Oligomeric Aq-477 may have been selected in hyperthermophiles because subunit association provides extra stabilization.

Insight into molecular stability and physiological properties of the diheme cytochrome CYC41 from the acidophilic bacterium Acidithiobacillus ferrooxidans.

The cyc1 gene encoding the soluble dihemic cytochrome c CYC(41) from Acidithiobacillus ferrooxidans, an acidophilic organism, has been cloned and expressed in Escherichia coli as the host organism. The cytochrome was successfully produced and folded only in fermentative conditions: this allowed us to determine the molecular basis of the heme insertion at extreme pH. Point mutations at two sequence positions (E121 and Y63) were introduced near the two hemes in order to assign individual redox potentials to the hemes and to identify the interaction sites with the redox partners, rusticyanin and cytochrome oxidase. Characterization of mutants E121A, Y63A, and Y63F CYC(41) with biochemical and biophysical techniques were carried out. Substitution of tyrosine 63 by phenylalanine alters the environment of heme B. This result indicates that heme B has the lower redox potential. Interaction studies with the two physiological partners indicate that CYC(41) functions as an electron wire between RCy and cytochrome oxidase. A specific glutamate residue (E121) located near heme A is directly involved in the interaction with RCy. A docking analysis of CYC(41), RCy, and cytochrome oxidase allowed us to propose a model for the complex in agreement with our experimental data.

Interaction and electron transfer between the high molecular weight cytochrome and cytochrome c3 from Desulfovibrio vulgaris Hildenborough: kinetic, microcalorimetric, EPR and electrochemical studies.

The complex formation between the tetraheme cytochrome c3 and hexadecaheme high molecular weight cytochrome c (Hmc), the structure of which has recently been resolved, has been characterized by cross-linking experiments, EPR, electrochemistry and kinetic analysis, and some key parameters of the interaction were determined. The analysis of electron transfer between [Fe] hydrogenase, cytochrome c3 and Hmc demonstrates a redox-shuttling role of cytochrome c3 in the pathway from hydrogenase to Hmc, and shows an effect of redox state on the interaction between the two cytochromes. The role of polyheme cytochromes in electron transfer from periplasmic hydrogenase to membrane redox proteins is assessed. A model with cytochrome c3 as an intermediate between hydrogenase and various polyheme cytochromes is proposed and its physiological consequences are discussed.

Voltammetry of a "protein on a rope".

A periplasmic electron-transfer protein, cytochrome c(555)(m) from Aquifex aeolicus contains a 62-residue N-terminal extension by which it is anchored to the membrane--most probably via a thioester bond to its N-terminal cysteine. This linker can act as a "rope" to tether the protein close to its reaction partners. Mimicking this principle, a recombinant cytochrome c(555)(m), expressed in Escherichia coli, has been attached covalently to a gold electrode modified with 6-mercaptohexan-1-ol. The "tethered" cytochrome c(555)(m) displays remarkably fast electron-transfer kinetics, with an electrochemical exchange rate constant k(0) of 1.4 x 10(4) s(-1). The results show that fast electron transfer is associated with weak interactions: importantly, the tethered cytochrome can explore many different orientations without escaping into solution.