Cytochrome aa³ from Sulfolobus acidocaldarius

Abstract

The thermoacidophilic archaebacterium Sulfolobus acidocaldurius (DSM 639) extrudes protons when expending respiratory energy [Moll, R. & Schiifer, G. (1988) FEBS Lett. 232, 359 -3631. Cytochromes of the membrane electron-transport systems are assumed to represent the proton pumps. Only a-and b-type cytochromes can be found; no c-type cytochromes are present. Of the two terminal oxidases [Anemuller, S. & Schafer, G. (1989) FEBS Lett. 244,451 -4551 one shows an absorption band at 604-605 nm, typical of cytochromes of the au3 type. This hemoprotein has been solubilized from the membrane and purified to homogeneity. It exhibits distinct differences from known ua3-type oxidases. (a) It consists of a single polypeptide subunit of 38 -40 kDa apparent molecular mass with two heme-a molecules and two copper ions. (b) In the oxidized state, absorption maxima are found at 421 nm and 597 nm, and in the reduced state at 439 nm and 601 nm; CO difference spectra suggest one heme to be a heme-ag centre. (c) The redox potentials of the heme centres are + 220 mV and + 370 mV, respectively. (d) A high-spin heme signal at g = 6 is present in EPR spectra, which is more prominent than the low-spin heme signal at g = 3, the former already being present in the oxidized state. A signal at g = 2.1 may be due to one of the copper ions and is superimposed upon a minor free radical signal at g = 2. (e) Caldariella quinone was also isolated from the plasma membrane of Sulfolobus. Its redox midpoint potential at pH 6.5 was determined to be + 100 ( & 5) mV; spectral properties have also been determined. (0 The isolated aa3 preparation does not oxidize cytochrome c; however, it oxidizes N,N,N,N-tetramethyl-l,4-phenylenediamine dihydrochloride as an artificial single-electron donor as well as reduced caldariella quinone, which is assumed to represent the natural substrate. The reaction is cyanide-sensitive and the product of oxygen reduction is water. (8) On the basis of the results obtained a novel type of cytochrome uu3 is postulated in this paper which oxidizes reduced quinones; its ability to act as a proton pump remains to be shown.Terminal oxidases from aerobic archaebacteria have not yet been studied in much detail, although they are of interest from two points of view: (a) their phylogenetic relation to eubacteria and eucaryotes as the two other evolutionary kingdoms; (b) their adaptation to extreme conditions, as in the case of the thermoacidophile Suljolohus acidocaldurius which grows at 75 -80" C and between pH 2 and 3. This bacterium has been shown to generate a proton-motive force of about 180 mV, thus maintaining its internal pH close to neutrality. The membrane electron-transport system consists of an NADH dehydrogenase [4], a succinate dehydrogenase and a special sulfur-containing quinone analogue, caldariella quinone [5]. Spectroscopic studies indicate that only a-and htype cytochromes are present [2,6], whereas cytochrome c and a be, complex are absent. This simple respiratory system is likely to contain two terminal oxidases...

EPR studies of cytochrome aa3 from Sulfolobus acidocaldarius

“…The interaction potential obtained is similar to that found for the E. coli cytochrome bo [25]. However, the intrinsic heme reduction potentials are different, reflecting either the different heme structures or the different reduction potentials of the natural reductants: menaquinone has Eo = 0 mV, while caldariella quinone has Eo = 100 mV [5]. The reduction potentials obtained through the EPR redox titration are different from those previously presented, obtained from a visible redox titration [4].…”

“…Cultivation of Sulfolobus acidocaldarius (DSM 639), membrane preparation and purification of cytochrome aa3 were done as described previously [5]. Membrane protein was determined as in [XI and detergent-solubilized protein was determined by the Lowry method in the presence of SDS [9].…”

“…N,N,N',N'-Tetramethyl-l,4-phenylenediamine dihydrochloride [Ph(NMeJ2] oxidation was followed as described previously at 546 nm [5]. Heme A content was determined using E~~~~~ (reduced -oxidized) = 12 mM-' cm- [lo].…”

“…The resonance Raman spectra of the oxidized enzyme are similar to those of the mammalian and plant cytochrome aa3, but the spectrum of the reduced form differs remarkably: in contrast to the beef heart enzyme, the formyl vibrations of heme a ruled out significant hydrogen bonding interactions. Preliminary EPR studies confirmed the presence of highspin and low-spin heme centers [5]. However, both heme centers were not quantitated and their redox behaviour was not studied.…”

“…Anemiiller Optical spectra clearly indicated the presence of two different heme centers with apparent redox potentials of + 220 mV and f370 mV at pH = 7.4 [5]. One of the hemes binds CO and therefore was identified as heme a3.…”