The frequency of IVH could be reduced significantly, if extremely premature infants, the vast majority of patients suffering from IVH, did not have to be transferred postnatally to another hospital.

The thermoacidophilic archaeon Sulfolobus acidocaldarius expresses a very unusual quinol oxidase, which contains four heme a redox centers and one copper atom. The enzyme was solubilized with dodecyl maltoside and purified to homogeneity by a combination of hydrophobic interaction and anion exchange chromatography. The oxidase complex consists of four polypeptide subunits with apparent molecular masses of 64, 39, 27, and 14 kDa that are encoded by the soxABCD operon (Lü bben, M., Kolmerer, B., and Saraste, M. (1992) EMBO J. 11, 805-812). The optical spectra and redox potentials of the SoxABCD complex have been characterized, and the absorption coefficients of the contributing cytochromes a 587 and aa 3 were determined. The EPR spectra indicate the presence of three low spin and one high spin heme species, the latter associated with the binuclear heme Cu B site. Standard midpoint potentials of the cytochrome a 587 heme centers were determined as ؉210 and ؉270 mV, respectively. The maximum turnover of the complex (1300 s ؊1 at 65°C) was found to be about three times greater than that of the previously studied isolated cytochrome aa 3 subunit alone (Gleissner, ؉ /e ؊ ratio >1 was determined, identifying the SoxABCD complex as a proton-pumping quinol oxidase. According to structural analysis, the cytochrome aa 3 moiety of the complex does not contain the signature of a H ؉ pumping channel as identified in Rhodobacter sphaeroides or Paracoccus denitrificans. Therefore, for H ؉ translocation, a mechanism different from that in typical heme-copper oxidases of the aa 3 or bo 3 type is discussed.M

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Extremophilic organisms are adapted to harsh environmental conditions like high temperature, extremely acidic or alkaline pH, high salt, or a combination of those. With a few exceptions extremophilic bacteria are colonizing only moderately hot biotopes, whereas hyperthermophiles are found specifically among archaea (formerly 'archaebacteria') which can thrive at temperatures close to or even above the boiling point of water. It has been a challenging question whether the special properties of their proteins and membranes have been acquired by adaptation, or whether they might reflect early evolutionary states as suggested by their phylogenetic position at the lowest branches of the universal tree of life.

The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermoacidophilic archaeon Suljolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S. acidocaldarius. With the electron donor system ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50°C. Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady-state transmembrane electrical potential ( A Y' , interior negative), and transmembrane pH gradient (ApH, interior alkaline) at temperatures up to 70°C. The successful functional reconstitution of the cytochrome aa,-type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon.The study of the bioenergetic system of aerobic, extremely thermoacidophilic archaea is of special interest for three reasons. First, archaea appeared very early in evolution as anaerobic species and were harshly exposed to the drastic change in the atmosphere from reducing to oxidizing conditions. Secondly, the evolutionary rates in the archaeal kingdom are very low suggesting the presence of still very simple structures of the energy-converting system. Thirdly, the drastic thermal and acidic conditions in their habitat imply very stable structures for their proteins and membrane architectureThe terminal quinol oxidase of the thermoacidophilic archaeon Suljolobus ucidocaldarius has been studied extensively by various biophysical methods. This enzyme was the first to be shown to react as a quinol oxidase of the cytochrome aa, type [4]. Subsequently, bacterial cytochrome ua3 was also found to act as a quinol oxidase 151. Biophysical characterization by EPR spectroscopy indicated a high-spin and a low-spin heme center, which could be attributed to the heme a, and heme a moieties. In addition, the presence of a binuclear center, composed of heme a3 and copper B could be demonstrated [6]. Furthermore, resonance-Raman spectroscopy suggested an unusual heme environment in the enzyme showing the lack of significant hydrogen bonds for the Correspondence to

Immunoisolation of hybrid liver support systems (LSS) utilizing suitable semipermeable membranes as an immune barrier enables neither immunocompetent cytotoxic factors to cause damage to the hepatocytes in the bioreactor nor xenogenic hepatocyte products to cause immunological side effects in patients. To determine the capability of membranes as an immune barrier, 6 flat membranes were investigated: Cuprophan (C-100), cut-off MW 1000, Cuprophan (C-240), cut-off MW 10,000, Polypropylen hydrophilic and hydrophobic (PPhi, PPho), cut-off MW 500,000-1,000,000, Polysulfon (PS), cut-off MW 1,000,000, Polyamid (PA), cut-off beyond MW 1,000,000. The permeability of the membranes to plasma factors and liver protein fractions (LP) was studied by routine biochemical methods and gel electrophoresis. In a second study, pigs (n=7) were immunised by LP after membrane passage. The results showed PA, PS, and PPhi to be completely permeable for plasma factors and LP C-100 and C-240 for urophanic substances, and C-240 again for LP under MW 14.000. All 7 pig sera studied by Western blot discovered pre-formed xenoreactive natural IgG-antibodies (NAB) against human liver antigen (AG) with MW 26.000. AB de-novo-synthesis was demonstrated for AG with MW 45.000. No AB-synthesis was induced for epitopes under MW 26,000. These results suggest that limiting the cut-off of bioreactor outflow membranes to MW < 26,000 could avoid immunological side effects to patients.

The integral quinol oxidase complex of Sulfolobus acidocaldarius (DSM 639) was investigated by resonance Raman spectroscopy. The complex includes four heme a groups which constitute two functional entities, a587 and aa3, containing two low-spin hemes and a low-spin as well as a high-spin heme, respectively. RR spectra were obtained from the fully oxidized and fully reduced states of the complex using different excitation wavelengths in the Soret band region in order to disentangle the contributions from the four heme groups. For the oxidized state, this approach allowed for the identification of two spectrally different types of heme a which were assigned to the bishistidine ligated hemes a of aa3 and a587 (type II) and to the additional heme a of a587 which is ligated by a histidine and methionine (type I). The spectra of both heme a types differ substantially from that of beef heart cytochrome c oxidase. In particular, the formyl stretching modes of types II and I are upshifted by 8 and 15 cm-1, respectively, implying a largely hydrophobic environment of the formyl groups in the quinol oxidase of Sulfolobus. Furthermore, the RR spectra of the oxidized state reveal the characteristic marker bands of a five-coordinated and a six-coordinated high-spin state, indicating that heme a3 exists in a coordination equilibrium, which is in sharp contrast to the purely six-coordinated high-spin configuration of heme a3 in any (quinol or cytochrome) oxidases studied so far. Also the formyl stretching mode of heme a3 appears to be unusual as its frequency is substantially lower than in beef heart oxidase. In the fully reduced state, no heterogeneity of heme a3 is observed and also the spectra of the various hemes a are nearly indistinguishable. Moreover, the formyl stretching vibrations of all hemes a and a3 apparently coincide to one prominent peak at 1658 cm-1 characteristic for a non-hydrogen-bonded carbonyl group. This finding is unique compared to other aa3 oxidases in which the formyl stretchings give rise to widely separated bands at approximately 1610 and approximately 1665 cm-1 for heme a and a3, respectively. In both the oxidized and the reduced states, the spectra of the aa3 entity in the integral complex differ significantly from those of the isolated aa3 entity studied previously [Heibel, G., Anzenbacher, P., Hildebrandt, P., & Schäfer, G. (1993a) Biochemistry 32, 10878-10884], indicating substantial interactions between the various subunits of the integral complex.

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