Irina Smirnova

Lactose permease and the alternating access mechanism.

Crystal structures of the lactose permease of Escherichia coli (LacY) reveal 12, mostly irregular transmembrane α-helices surrounding a large cavity open to the cytoplasm and a tightly sealed periplasmic side (inward-facing conformation) with the sugar-binding site at the apex of the cavity and inaccessible from the periplasm. However, LacY is highly dynamic, and binding of a galactopyranoside causes closing of the inward-facing cavity with opening of a complementary outward-facing cavity. Therefore, the coupled, electrogenic translocation of a sugar and a proton across the cytoplasmic membrane via LacY very likely involves a global conformational change that allows alternating access of sugar- and H(+)-binding sites to either side of the membrane. Here the various biochemical and biophysical approaches that provide strong support for the alternating access mechanism are reviewed. Evidence is also presented indicating that opening of the periplasmic cavity is probably the limiting step for binding and perhaps transport.

The sodium cycle. I. Na+-dependent motility and modes of membrane energization in the marine alkalotolerant vibrio Alginolyticus.

Respiration, membrane potential generation and motility of the marine alkalotolerant Vibrio alginolyticus were studied. Subbacterial vesicles competent in NADH oxidation and delta psi generation were obtained. The rate of NADH oxidation by the vesicles was stimulated by Na+ in a fashion specifically sensitive to submicromolar HQNO (2-heptyl-4-hydroxyquinoline N-oxide) concentrations. The same amounts of HQNO completely suppressed the delta psi generation. Delta psi was also inhibited by cyanide, gramicidin D and by CCCP + monensin. CCCP (carbonyl cyanide m-chlorophenylhydrazone) added without monensin exerted a much weaker effect on delta psi. Na+ was required to couple NADH oxidation with delta psi generation. These findings are in agreement with the data of Tokuda and Unemoto on Na+-motive NADH oxidase in V. alginolyticus. Motility of V. alginolyticus cells was shown to be (i) Na+-dependent, (ii) sensitive to CCCP + monensin combination, whereas CCCP and monensin, added separately, failed to paralyze the cells, (iii) sensitive to combined treatment by HQNO, cyanide or anaerobiosis and arsenate, whereas inhibition of respiration without arsenate resulted only in a partial suppression of motility. Artificially imposed delta pNa, i.e., addition of NaCl to the K+ -loaded cells paralyzed by HQNO + arsenate, was shown to initiate motility which persisted for several minutes. Monensin completely abolished the NaCl effect. Under the same conditions, respiration-supported motility was only slightly lowered by monensin. The artificially-imposed delta pH, i.e., acidification of the medium from pH 8.6 to 6.5 failed to activate motility. It is concluded that delta mu Na+ produced by (i) the respiratory chain and (ii) an arsenate-sensitive anaerobic mechanism (presumably by glycolysis + Na+ ATPase) can be consumed by an Na+ -motor responsible for motility of V. alginolyticus.

HOQNO interaction with cytochrome b in succinate:menaquinone oxidoreductase from Bacillus subtilis.

2‐n‐Heptyl4‐hydroxyquinoline‐N‐oxide (HOQNO) inhibits the succinate:quinone oxidoreductase activity of isolated and membrane‐bound succinate:menaquinone oxidoreductase of B. subtilis. The inhibition pattern resembles closely that observed for α‐thenoyltrifluoroacetone and carboxins in the mitochondrial succinate:ubiquinone oxidoreductase: ca. 90% of the activity is highly sensitive to HOQNO (K i ca. 0.2 μM for the isolated enzyme) whereas the rest 10% proves to be resistant to the inhibitor. HOQNO binding is shown to perturb the absorption spectrum of the ferrous di‐heme cytochrome b of the B. subtilis succinate:quinone oxidoreductase both in the α and Soret bands. In addition, the inhibitor is shown to bring about a negative shift of E m of the low‐potential heme b. It is suggested that HOQNO interacts with a menasemiquinone binding site near the low‐potential heme and suppresses the MQ.−‐to‐MQH2 step of the quinone reductase reaction but allows partly for the MQ‐to‐MQ.− transition to occur; dismutation of MQ. formed in the latter reaction to MQ and MQH2 may account for the 10% of the enzyme activity insensitive to HOQNO.

Probing of the rates of alternating access in LacY with Trp fluorescence

Sugar/H+ symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H+ binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pKa of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is ≈100 s−1. On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140→Trp/Phe-334→His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pKa = 8), but remarkably, the rate of sugar-induced quenching is only ≈0.4 s−1. The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins.

Opening the periplasmic cavity in lactose permease is the limiting step for sugar binding

The lactose permease (LacY) catalyzes galactoside/H+ symport via an alternating access mechanism in which sugar- and H+-binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by opening and closing of inward- and outward-facing cavities. The crystal structures of wild-type LacY, as well as accessibility data for the protein in the membrane, provide strong support for a conformation with a tightly closed periplasmic side and an open cytoplasmic side (an inward-facing conformation). In this study, rates of substrate binding were measured by stopped-flow with purified LacY either in detergent or in reconstituted proteoliposomes. Binding rates are compared with rates of sugar-induced opening of the periplasmic pathway obtained by using a recently developed method based on unquenching of Trp fluorescence. A linear dependence of galactoside-binding rates on sugar concentration is observed in detergent, whereas reconstituted LacY binds substrate at a slower rate that is independent of sugar concentration. Rates of opening of the periplasmic cavity with LacY in detergent are independent of substrate concentration and are essentially the same for different galactosidic sugars. The findings demonstrate clearly that reconstituted LacY is oriented physiologically with a closed periplasmic side that limits access of sugar to the binding site. Moreover, opening of the periplasmic cavity is the limiting factor for sugar binding with reconstituted LacY and may be the limiting step in the overall transport reaction.

Rapid kinetics of membrane potential generation by cytochrome c oxidase with the photoactive Ru(II)‐tris‐bipyridyl derivative of cytochrome c as electron donor

Yeast iso‐1‐cytochrome c covalently modified at cysteine‐102 with (4‐bromomethyl‐4′‐methylbipyridine)[bis(bi‐pyridine)]Ru2+ (Ru‐102‐Cyt c) has been used as a photoactive electron donor to mitochondrial cytochrome c oxidase (COX) reconstituted into phospholipid vesicles. Rapid kinetics of membrane potential generation by the enzyme following flash‐induced photoreduction of Ru‐102‐Cyt c heme has been measured and compared to photovoltaic responses observed with Ru(II)(bipy‐ridyl)3 (RuBpy) as the photoreductant [D.L. Zaslavsky et al. (1993) FEBS Lett. 336, 389–393]. At low ionic strength, when Ru‐102‐Cyt c forms a tight electrostatic complex with COX, flash‐activation results in a polyphasic electrogenic response corresponding to transfer of a negative charge to the interior of the vesicles. The initial rapid phase is virtually identical to the 50 μs transient observed in the presence of RuBpy as the photoactive electron donor which originates from electrogenic reduction of heme a by CuA. CuA reduction by Ru‐102‐Cyt c turns out to be not electrogenic in agreement with the peripheral location of visible copper in the enzyme. A millisecond phase (τ ca. 4 ms) following the 50 μs initial part of the response and associated with vectorial translocation of protons linked to oxygen intermediate interconversion in the binuclear centre, can be resolved both with RuBpy and Ru‐102‐Cyt c as electron donors; however, this phase is small in the absence of added H2O2. In addition to these two transients, the flash‐induced electrogenic response in the presence of Ru‐102‐Cyt c reveals a large slow phase of Δψ generation not observed with RuBpy. This phase is completely quenched upon inclusion of 100 μM ferricyanide in the medium and originates from a second order reaction of COX with the excess Ru‐102‐Cyt c 2+ generated by the flash in a solution.

Electron and proton transfer in the ba (3) oxidase from Thermus thermophilus.

The ba3-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa3–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O2 to water is catalysed at a haem a3-CuB catalytic site. The three-dimensional structure of the ba3 oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa3 oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba3-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k≅6.8 × 104 s−1) and formation of the peroxy (PR) intermediate. In the next step a proton was taken up from solution with a rate constant of ∼1.7 × 104 s−1, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ∼1,100 s−1, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the PR and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba3 oxidases, the proton-uptake reactions occur over the same time scales as in the aa3-type oxidases.

Delineating Electrogenic Reactions during Lactose/H+ Symport

Electrogenic reactions accompanying downhill lactose/H+ symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H+ translocation is abolished either by acidic pH or by a Glu325 → Ala mutation in the H+ binding site (this step with a rate constant of ∼200 s−1 for wild-type LacY leads to an intermediate proposed to represent an “occluded” state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of ∼30 s−1 and a pK of 7.5. This partial reaction is assigned to rate-limiting H+ release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical−biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway.

Role of protons in sugar binding to LacY

WT lactose permease of Escherichia coli (LacY) reconstituted into proteoliposomes loaded with a pH-sensitive fluorophore exhibits robust uphill H+ translocation coupled with downhill lactose transport. However, galactoside binding by mutants defective in lactose-induced H+ translocation is not accompanied by release of an H+ on the interior of the proteoliposomes. Because the pKa value for galactoside binding is ∼10.5, protonation of LacY likely precedes sugar binding at physiological pH. Consistently, purified WT LacY, as well as the mutants, binds substrate at pH 7.5–8.5 in detergent, but no change in ambient pH is observed, demonstrating directly that LacY already is protonated when sugar binds. However, a kinetic isotope effect (KIE) on the rate of binding is observed, indicating that deuterium substitution for protium affects an H+ transfer reaction within LacY that is associated with sugar binding. At neutral pH or pD, both the rate of sugar dissociation (koff) and the forward rate (kon) are slower in D2O than in H2O (KIE is ∼2), and, as a result, no change in affinity (Kd) is observed. Alkaline conditions enhance the effect of D2O on koff, the KIE increases to 3.6–4.0, and affinity for sugar increases compared with H2O. In contrast, LacY mutants that exhibit pH-independent high-affinity binding up to pH 11.0 (e.g., Glu325 → Gln) exhibit the same KIE (1.5–1.8) at neutral or alkaline pH (pD). Proton inventory studies exhibit a linear relationship between koff and D2O concentration at neutral and alkaline pH, indicating that internal transfer of a single H+ is involved in the KIE.

Cyanide-reactive sites in cytochrome bd complex from E. coli

Cyanide reacts with cytochrome bd from E. coli in an ‘aerobically oxidized’ state (mainly, an oxygenated complex b 558 3+ b 595 3+ d 2+‐O2), bringing about (i) decomposition of the heme d 2+ oxycomplex (decay of the 648 nm absorption band) and (ii) extensive red shift in the Soret region accompanied by minor changes in the visible range assigned to ferric heme b 595. MCD spectra show that the Soret red shift is associated with heme b 558 3+ high‐to low‐spin transition. This is the first unambiguous demonstration that heme b 595 can bind exogenous ligands. No reaction of cyanide with b 558 is observed. In about 70% of the enzyme which forms the cyano complex, the spin‐state transition of b 595 decay of heme d oxycomplex match each other kinetically (k eff ca. 0.002 s−1 at 50 mM KCN, pH 8.1, 25°C). This points to an interaction between the two hemes. The concerted binding of cyanide to d 3+ and b 595 3+, perhaps as a bridging ligand, is probably rate‐limited by d 2+ oxycomplex autoxidation. In the remaining 30% of the isolated bd, there is a rapid phase of cyanide‐induced b 595 spin‐state transition which can be tentatively assigned to that proportion of the enzyme in which heme d is initially in the ferric rather than ferrous‐oxy form.