Characterization and application of a thermostable primary transport system: Cytochrome‐C oxidase from Bacillus stearothermophilus

Abstract

Cytochrome-c oxidase from Bacillus stearothermophilus has been purified to homogeneity by detergent extraction followed by DEAE-cellulose, hydroxyapatite-and gel-filtration chromatography. The enzyme is a typical cytochrome-aa3-type oxidase which binds carbon monoxfde and is sensitive to classical oxidase inhibitors like cyanide and azide. The purified enzyme is composed of three different subunits (57, 37 and 22 kDa). The subunit with intermediate molecular mass contains a covalently attached heme-c moiety. The enzyme appeared to be extremely thermostable (inactivation temperature = 81 "C). Highest turnover rates of the reconstituted enzyme were obtained with Saccharomyces cerevisiae cytochrome c or reduced forms of non-physiological electron donors like N,N,N',N'-tetramethyl-p-phenylenediamine and phenazine methosulphate. The reconstituted enzyme can generate a proton-motive force consisting of a high membrane potential and trans-membrane pH gradient. The high electro-motive force of the enzyme (dp = -180 to -200 mV) indicates that this enzyme functions as a high-capacity electrogenic proton pump.Liposomes containing the purified thermostable and thermoactive cytochrome-c oxidase were fused with membranes from the fermentative bacterium Clostridium acetobutylicurn. In the hybrid system a high protonmotive force can be generated upon oxidation of reduced N,N,N,N'-tetramethyl-p-phenylenediamine by the incorporated oxidase which subsequently can be used to drive secondary transport of amino acids. This demonstrates the applicability of the cytochrome-c oxidase to study solute transport in membranes of fermentative bacteria.Bacterial cytoplasmic membranes are equipped with energy-transducing enzymes, capable of converting redox, chemical or light-energy into electrochemical energy [l -31. The electrochemical energy generated by these enzymes fulfills an essential role in many bioenergetic processes [4].In the past decade several bacterial membrane-bound energy-converting enzymes have been isolated and extensively characterized [5 -171. The structural simplicity of these bacterial enzymes in comparison to their eukaryotic counterparts offers attractive possibilities to study the molecular mechanism(s) of energy transduction. Energy-converting enzymes from thermophilic bacteria possess an additional attractive feature, i. e. their enhanced (therm0)stability [18]. This is especially convenient in the purification and the utilization of these enzymes. Some membrane-bound energy-converting enzymes (cytochrome-c oxidases) from thermophilic bacteria, like Thermus thermophilus HB-8 and Bacillus PS3 have already Correspondence to W . N . Konings, Laboratorium voor Microbiologie, Rijksuniversiteit Groningen, Biologisch Centrum,

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus

“…Since the proton motive force was found to be a major driving force for amino acid transport, the influence of the medium composition on the Ap was studied. In previous publications de Vrij et al (9,10) demonstrated that in potassium phosphate (pH 6.0), oxidation via the respiratory chain of the electron donor ascorbate-TMPD results, in B. stearothermophilus membrane vesicles, in the generation of a high membrane potential (Aj = -105 mV) and a pH gradient (-ZA&pH = -45 mV). Qualitative information about the membrane potential generated has been obtained with the indicator diSC (3) to an interconversion of membrane potential into a pH gradient.…”

“…Uptake of L-alanine (2.87 ,uM) or L-leucine (1.43 I1M) was measured at 45°C in oxygen-saturated 50 mM MES-NaOH (pH 6.0) containing 10 mM MgSO4. The figure shows L-alanine and L-leucine uptake in the absence of electron donors (0), in the presence of the electron donors ascorbate (20 mM) and TMPD (300 ,uM) without ionophores (0), and in the presence of monensin (20 nM) (L) or nonactin (10 membrane (ApH; interior alkaline) were determined at 45°C from the fluorescence changes of pyranine (excitation at 460 nm and emission at 508 nm [7,8] (20 mM) and TMPD (100 to 300 FuM). Monensin and nonactin were used at the final concentrations given above.…”

Thermal Stability of the Human Immunodeficiency Virus Type 1 (HIV-1) Receptors, CD4 and CXCR4, Reconstituted in Proteoliposomes

“…The inactivation temperature (T i ) can be defined as the temperature when the half-life t 1/2 of thermal denaturation is equal to 10 minutes [80], [82], [126]. The value of T i can be found by regression analysis of the line when the log 10 (t 1/2 ) values versus temperature are plotted.…”

A fourth subunit is present in cytochrome c oxidase from the thermophilic bacterium PS3

“…Similar aa3-type oxidases with 3 subunits have been purified from Bacillus subtilis [4]. Bacillus stearothermophilus [5], Bacillus firmus [6] and Paracoccus denitrificans [7]. As the original preparation of the Paracoccus envme consists of two subunits [8,9], the reported two-subunit aa3-type oxidases of bacteria (see [ 10,l I] for reviews) may have lost the third subunit.…”