We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodiumproton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

F 1 F 0 ATP synthases generate ATP by a rotary catalytic mechanism in which H + transport is coupled to rotation of an oligomeric ring of c subunits extending through the membrane. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Subunit a of the F 0 sector is thought to provide proton access channels to and from aspartyl-61. Here, we summarize new information on the structural organization of Escherichia coli subunit a and the mapping of aqueous-accessible residues in the second, fourth and ¢fth transmembrane helices (TMHs). Aqueous-accessible regions of these helices extend to both the cytoplasmic and periplasmic surface. We propose that aTMH4 rotates to alternately expose the periplasmic or cytoplasmic half-channels to aspartyl-61 of subunit c during the proton transport cycle. The concerted rotation of interacting helices in subunit a and subunit c is proposed to be the mechanical force driving rotation of the c-rotor, using a mechanism akin to meshed gears.

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The role of subunit a in promoting proton translocation and rotary motion in the Escherichia coli F 1Fo ATP synthase is poorly understood. In the membrane-bound F o sector of the enzyme, H ؉ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp-61 at the center of the membrane via aqueous channels formed at least in part by one or more of the five TMHs of subunit a. Aqueous access pathways have previously been mapped to surfaces of aTMH4. Here we have substituted Cys into the second and fifth TMHs of subunit a and carried out chemical modification with Ag ؉ and N-ethylmaleimide to define the aqueous accessibility of residues along these helices. Access to cAsp-61 at the center of the membrane may be mediated in part by Ag ؉ -sensitive residues 248, 249, 251, and 252 in aTMH5. From the periplasmic surface, aqueous access to cAsp-61 may be mediated by silver-sensitive residues 115, 116, 119, 120, 122, and 126 in aTMH2. The Ag ؉ -sensitive residues in TMH2, -4, and -5 form a continuum extending from the periplasmic to the cytoplasmic side of the membrane. In an arrangement of helices supported by second-site revertant and crosslinking analyses, these residues cluster at the interior of a four-helix bundle formed by TMH2-5. The aqueous access pathways at the interior of subunit a may be gated by a swiveling of helices in this bundle, alternately exposing cytoplasmic and periplasmic half channels to cAsp-61 during the H ؉ transport cycle. H ϩ transporting F 1 F o ATP synthases consist of two structurally and functionally distinct sectors termed F 1 and F o . In the intact enzyme, ATP synthesis or hydrolysis takes place in the F 1 sector and is coupled to active H ϩ transport through the F o sector. Structurally similar F 1 F o ATP synthases are present in mitochondria, chloroplasts, and most eubacteria (1). The F 1 sector lies at the surface of the membrane and in Escherichia coli consists of five subunits in an ␣ 3 ␤ 3 ␥ 1 ␦ 1 1 stoichiometry. The F o sector spans the membrane and in E. coli consists of three subunits in an a 1 b 2 c 10 stoichiometry (2). In the complete membranous enzyme, the rotation of subunit ␥ is proposed to be driven by H ϩ transport-coupled rotation of a connected ring of c subunits in the F o sector of the enzyme. The c subunit spans the membrane as a hairpin of two ␣-helices, and in the case of E. coli, contains the essential Asp-61 residue at the center of the second transmembrane helix (TMH). Asp-61 is thought to undergo protonation and deprotonation as each subunit of the oligomeric ring moves past a stationary subunit a. Subunit a is believed to provide access channels to the proton-binding Asp-61 residue, but the actual proton translocation pathway is only partially defined (3-5).Subunit a is known to fold with five TMHs (6-8), with aTMH4 packing in parallel to cTMH2, i.e., the helix to which Asp-61 is anchored (9). The interaction of the conserved Arg-210 residue in aTMH4 with cTMH2 is thought to be cri...

F(1)F(o) ATP synthases generate ATP by a rotary catalytic mechanism in which H(+) transport is coupled to rotation of a ring of c subunits within the transmembrane sector of the enzyme. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Proton access channels to and from aspartyl-61 are thought to form in subunit a of the F(o) sector. Here, we summarize new information on the structural organization of subunit a and the mapping of aqueous accessible residues in the fourth and fifth transmembrane helices (TMHs). Cysteine substituted residues, lying on opposite faces of aTMH-4, preferentially react with either N-ethyl-maleimide (NEM) or Ag(+). We propose that aTMH-4 rotates to alternately expose each helical face to aspartyl-61 of subunit c during the proton transport cycle. The concerted helical rotation of aTMH-4 and cTMH-2 are proposed to be coupled to the stepwise mechanical movement of the c-rotor.