Grown under anaerobic conditions in presence of nitrate, Haloferax volcanii shows nitrate reduction and accumulation of nitrite in the culture medium. We found a membrane-associated nitrate reductase, which could easily be solubilized by gently stirring of isolated membranes. Surprisingly, this nitrate reductase requires no NaCl for its activity. A medium pH of 7.5 and high temperatures up to 80 °C are necessary for optimum activity. Kinetic studies showed that the apparent Kᴍ was 0.36 mmol/l for nitrate and 80 μmol/l for dithionite-reduced methyl viologen. The respiratory chain inhibitor cyanide effects nitrate reduction noncompe-titively with respect to nitrate with a Ki of 0.3 mmol/l. Azide was a strong inhibitor: The concentration required for half maximal inhibition was 60 μmol/l, whereas thiocyanate and chlorate were much weaker inhibitors. The isolated enzyme was partially purified by frac­tionated precipitation using polyethylene glycol. SDS gel electrophoresis resulted in three putative subunits of the nitrate reductase of molecular masses of about 100, 61, and 31 kDa.

Both, Haloferax mediterranei and Haloferax volcanii membranes contain ATPases which are capable of hydrolyzing ATP in presence of Mg2+ or Mn2+. The ATPases require high concentrations of NaCl, a pH value of 9, and high temperatures up to 60 °C. Free manganese ions inhibited the enzyme activity of either ATPase. The ATPases of Hf. mediterranei and Hf. volcanii, respectively, show different sensitivities to inhibitors of ATP hydrolysis. ATP hydrolysis of isolated Hf. mediterranei ATPase was inhibited by NaN3, which was reported to be specific for F-ATPases, by nitrate and N-ethylmaleimide (NEM), which are specific inhibitors of V-ATPases. ATP hydrolysis of Haloferax mediterranei membranes was not inhibited by DCCD , but [14C]DCCD was bound to a 14 kDa peptide of the isolated, partially purified enzyme. Furthermore, the ATPase was inactivated by preincubation with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The ATPase activity of Hf. volcanii membranes was inhibited by NEM but not by nitrate and NaN3. SDS gel electrophoresis of the partially purified enzyme of Haloferax mediterranei showed putative ATPase subunits of 53.5, 49, 42, 22, 21, 14, 12, and 7.5 kDa. Immunoblots showed cross reactivity between a 53 kDa peptide and anti-β (chloroplast F1), as well as between 53, 50 and 47 kDa peptides and an ATPase antibody of Methanosarcina barkeri. The results will be discussed in context with the placement of the archaebacterial ATPases (A-ATPases) between F- and V-ATPases

The archaeal ATPase of the halophile Haloferax volcanii synthesizes ATP at the expense of a proton gradient, as shown by sensitivity to the uncoupler carboxyl cyanide p-trifluoromethoxyphenylhydrazone, to the ionophore nigericin, and to the proton channel-modifying reagent N,N-dicyclohexylcarbodiimide. The conditions for an optimally active ATP synthase have been determined. We were able to purify the enzyme complex and to identify the larger subunits with antisera raised against synthetic peptides. To identify additional subunits of this enzyme complex, we cloned and sequenced a gene cluster encoding five hydrophilic subunits of the A 1 part of the proton-translocating archaeal ATP synthase. Initiation, termination, and ribosome-binding sequences as well as the result of a single transcript suggest that the ATPase genes are organized in an operon. The calculated molecular masses of the deduced gene products are 22.0 kDa (subunit D), 38.7 kDa (subunit C), 11.6 kDa (subunit E), 52.0 kDa (subunit B), and 64.5 kDa (subunit A). The described operon contains genes in the order D, C, E, B, and A; it contains no gene for the hydrophobic, so-called proteolipid (subunit c, the proton-conducting subunit of the A 0 part). This subunit has been isolated and purified; its molecular mass as deduced by SDS-polyacrylamide gel electrophoresis is 9.7 kDa.The archaeon (archaebacterium) Haloferax volcanii belongs to the more moderate halophilic archaea, growing best in a medium containing 2 mol/liter NaCl and 0.25 mol/liter MgCl 2 at pH 7 (1). The proton-conducting ATPase of this archaeon is a halophilic enzyme that shows maximum activity at 1.75 mol/liter NaCl. It was characterized recently with respect to its hydrolytic function (2). The membrane-bound ATPases from archaea share properties with both bacterial (formerly eubacterial) and eucaryal (formerly eukaryotic) ATPases. Like F 0 F 1 -ATPases, they are able to synthesize ATP from ADP and phosphate coupled to a proton flow. Genetically derived amino acid sequences of the large subunits of archaeal ATPases (2-6), immunological cross-reactivity (7,8), and inhibition of the enzymes by specific inhibitors (1, 9 -11) deliver the main arguments for the conclusion that the archaeal ATPases resemble eucaryal vacuolar ATPases (V-ATPases) 1 more closely than bacterial F-ATPases.The primary structures of subunits A and B of H. volcanii are closely related to those of Halobacterium salinarium (halobium) (6) and indeed more related to the class of the eucaryal V-ATPases. On the other hand, the function of eucaryal VATPases is to generate a ⌬ H ϩ at the expense of ATP hydrolysis; they are not able to synthesize ATP in a reversed reaction. More detailed investigations on the intermediate position of different archaeal ATPases are required.Up to now, almost nothing was known about the small subunits of halophilic ATPases. F-ATPases have one copy each of three different small subunits, ␥, ␦, and ⑀, which are involved in proton conduction and/or regulation (for review, see Refs. 12 and 13). V-A...

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