Yasuo Sugiyama

AUSTRALIAN Halobacteria and THEIR RETINAL‐PROTEIN ION PUMPS*

Abstract– Halophiles collected in Western Australia have been found to be examples of extremely halophilic rod‐shaped archaebacteria, members of the genus Halobacterium. Most of them contain retinal proteins, and these proteins differ from one another and also from both bacteriorhodopsin (bR) and halorhodopsin [and sensory rhodopsins (sR)] isolated from Halobacterium salinarium (halobium), as revealed by their peptide maps and amino acid sequences. However, these retinal proteins still have the ability to pump protons or chloride ions in the light. These new ion pumps, designated archaerhodopsins (aR) [Mukohata et al. (1988) Biochem. Biophys. Res. Commun.151,1339–1345], are almost identical in terms of their molecular sizes and transient photochemical properties to the ion pumps identified previously. Differences are found in the: (1) apparent extinction coefficient of dark/light‐adapted aR‐2; (2) titration profiles at acidic pH of the absorption spectra of all aRs; and (3) circular dichroism spectra, which are influenced by the coexistent isoprenoid bacterioruberin. The amino acid sequences of two proton pumps from the Australian halobacteria, namely aR and aR‐2, are approximately 90% homologous and both sequences are about 60% homologous with that of bR. Hydropathy plots suggest that these pumps also have a seven‐helical structure similar to that of bR. The amino acid residues are highly conserved in the helical regions, in particular in the case of helices C and G (91 and 84%, respectively), among the three proton pumps.

Archaerhodopsin-2, from Halobacterium sp. aus-2 further reveals essential amino acid residues for light-driven proton pumps☆

We have isolated a retinal protein which differs from bacteriorhodopsin and archaerhodopsin and pumps out as many protons in the light as those proton pumps. We tentatively named it archaerhodopsin-2. We have cloned and sequenced the gene that encodes archaerhodopsin-2. The gene consists of 780-bp nucleotides for 259 amino acids with a molecular mass of 27,937 Da. The amino acid sequence of archaerhodopsin-2 is 56% identical to bacteriorhodopsin and 88% to archaerhodopsin, with a few gaps of a few amino acids in both cases. Although the amino acid sequence of archaerhodopsin has revealed 157 conserved residues common to bacteriorhodopsin, the sequence of archaerhodopsin-2 reduces that number to 133. Of these, 38 amino acids are also common to chloride pumps and 24 to all bacterial retinal proteins known to date.

The halobacterial H+-translocating ATP synthase relates to the eukaryotic anion-sensitive H+-ATPase.

The H+-translocating ATP synthase of Halobacterium halobium (Y. Mukohata and M. Yoshida (1987) J. Biochem. 102, 797-802) includes a catalytic moiety of 320 kDa which is isolated as an azide-insensitive ATPase (T. Nanba and Y. Mukohata (1987) J. Biochem. 102, 591-598). The polyclonal antibody against this archaebacterial ATPase cross-reacts with the anion-sensitive H+-ATPase of red beet, Beta vulgaris, tonoplast as well as with another archaebacterial ATPase from Sulfolobus acidocaldarius. The affinity is much higher than to F1-ATPase from spinach chloroplasts or to Ca2+-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle.

Modification of one lysine by pyridoxal phosphate completely inactivates chloroplast coupling factor 1 ATPase.

Chemical modification of chloroplast coupling factor 1 (CF,) in situ caused energy-transfer inhibition [l-3] of photophosphorylation and revealed essential arginyl [ 1,4] and lysyl [2,3] residues. When CF 1 in situ was modified by pyridoxal phosphate (PLP) [3] , the a, fl and y subunits were equally labeled and phosphorylation was inhibited. For 50% inhibition, 1 mol PLP was found in each of these 3 subunit fractions per mol CF 1 _ Since CFr has an allotopic nature [5] , it was of interest to examine PLP modification on isolated CFr , which was thus isolated from thylakoids, modified by PLP, then assayed for the heat-activated Ca”-ATPa.se [6] . It was found that modification of only 1 lysyl residue/CFr inactivated the Ca*‘-ATPase completely. For 50% inhibition, 0.5 mol PLP was found in each of the (Y and /3 subunit fractions per mol CFr , but none in the y subunit fraction.

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R1mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all‐trans‐retinal converted R1mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light‐dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R1mW. Electron micrographs of freeze‐fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure.

Cytochrome aa3 in Haloferax volcanii

A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN− complex spectrum that indicates the presence of heme a and heme a3. This cytochrome aa3 consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa3, providing physiological evidence for electron transfer from cytochrome c to cytochrome aa3 in archaea.

Energy transfer inhibition induced by modification of membrane-bound chloroplast coupling factor 1 by pyridoxal phosphate

The function of the live different subunits [l] of chloroplast coupling factor 1 (CF,) has been under active investigation. The modification of isolated CFr by 7-chloro4-nitrobenzo-2-oxa-l,3-diazole [2] suggests the involvement of tyrosyl residues in the active sites on the @ubunits. The modifications by butane2,3-dione [3,4] and fluorescamine [5] suggest the presence of essential arginyl and lysyl residue, respectively. Recently, pyridoxal phosphate (PLP) was used to detect active site lysyl residues in various proteins [6-81 which interact with phosphate-containing ligands. In this paper, we report that the modification of membrane-bound CFr by PLP inhibits phosphorylation without damaging electron transport, suggesting the participation of lysyl residue(s) in the activity of CFr .

Structural analysis of actinidin and a comparison of cadmium and sulfur anomalous signals from actinidin crystals measured using in-house copper- and chromium-anode X-ray sources.

The structure of the 24 kDa cysteine protease saru-actinidin from the fruit of Actinidia arguta Planch. (sarunashi) was determined by the cadmium/sulfur-SAD method with X-ray diffraction data collected using in-house Cu Kα and Cr Kα radiation. The anomalous scatterers included nine sulfurs and several cadmium ions from the crystallization solution. The high quality of the diffraction data, the use of chromium-anode X-ray radiation and the substantial anomalous signal allowed structure determination and automated model building despite both a low solvent content (<40%) and low data multiplicity. The amino-acid sequence of saru-actinidin was deduced from the cDNA and was modified based on experimental electron-density maps at 1.5 Å resolution. The active site of saru-actinidin is occupied by a cadmium ion and the active-site cysteine is found to be in an unmodified, cysteine sulfenic acid or cysteine sulfinic acid form. The cadmium sites, coordination geometries and polygonal water structures on the protein surface have also been extensively analyzed. An analysis and comparison of the sulfur/cadmium anomalous signals at the Cu Kα and Cr Kα wavelengths was carried out. It is proposed that the inclusion of cadmium salts in crystallization solutions coupled with chromium-anode radiation can provide a convenient route for structure determination.

Regulation of electron transport in isolated chloroplasts by sequential binding of adenine nucleotides to the coupling factor protein

Changes in the ferricyanide-reducing activity in isolated spinach chloroplasts by adenine nucleotides were measured in the presence or absence of phosphate, arsenate or pyrophosphate at 15°C. The activity changes were analyzed and ascribed to the interaction between the nucleotides and the chloroplast coupling factor (CF1).ADP and ATP (but not AMP) partially inhibited ferricyanide reduction through a 1∶1 binding to the inhibition site on CF1.When the ferricyanide reduction was coupled to either phosphorylation or arsenylation, the inhibition by ADP was released through a 1∶1 binding of the second ADP molecule to the coupling site on the CF1 with which the first ADP had been associated.The association constants of ADP for the inhibition and the coupling site were found to be approximately 5×105 and 6×104 M−1, respectively. TheKm value of ADP for arsenylation (pH 8.3) was around 17 μM.The ADP-regulated electron transport was defined based on these results. The ADP-regulated ferricyanide reduction, when coupled with phosphorylation, revealed a stoichiometry of P/Δe=1 between the amounts of esterified phosphate and reduced ferricyanide.

The light-driven proton pump, cruxrhodopsin-2 in Haloarcula sp. arg-2 (bR+, hR-), and its coupled ATP formation.

Haloarcula sp. arg-2, a natural bacterial isolate from Andes heights, has a light-driven proton pump but not a light-driven anion pump. We have cloned and sequenced the gene encoding for the proton pump which has been named cruxrhodopsin-2. The gene consists of 768 bp encoding 255 amino acids with a molecular mass of 27,544 Da. The deduced amino acid sequence of cruxrhodopsin-2 is 77%, 50%, 48% and 48% identical to those of cruxrhodopsin-1, bacteriorhodopsin, archaerhodopsin-1 and archaerhodopsin-2, respectively. The charged amino acids important for the proton pump function were conserved among all these molecules. Cruxrhodopsin-2 accounted for 0.05 nmol/mg protein in arg-2, which was 20-30-fold less than the proportion of bacteriorhodopsin in Halobacterium salinarium R1M1. In contrast to R1M1, under anaerobic conditions, arg-2 showed light-induced proton extrusion concomitant with an increase in ATP level without transient proton uptake. Dicyclohexylcarbodiimide enhanced the rate and extent of proton extrusion and inhibited ATP formation in the light. The apparent stoichiometry of H+/ATP was estimated to be more than three in this natural bR+hR- strain.