The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K þ or Na þ efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K þ to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH in ) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH in first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH in is achieved.antiporter | liposomes | M2 channel | protons | ion channel T he influenza A virus M2 protein is a tetrameric integral membrane protein containing a short N-terminal extracellular domain, a transmembrane (TM) helix, and a 54-residue cytoplasmic tail (1, 2). Four M2 TM domains associate into a highly selective proton channel (3, 4) whose activity is essential for viral replication (5, 6). Influenza virus enters cells via the endosomal pathway, and the M2 protein functions to equilibrate the pH of the virus interior with that of the acidic endosome. The lowering of the pH within the virus leads to disruption of the interactions between the viral ribonucleoprotein (RNP) complex and the M1 protein (5), an important step in viral uncoating (7). Additionally, for some subtypes of influenza A virus, the M2 proton channel activity helps maintain a neutral pH in the lumen of the trans-Golgi network to prevent premature triggering of the hemagglutinin to the low-pH form (8-11). A long-standing question has been how M2 can conduct a substantial number of protons into the very small-volume interior of the virus without developing a substantial electrical gradient. Diffusion of a few protons down their concentration gradient from the acid environment of the endosome into the viral interior would result in an electrical gradient that would abruptly halt further proton flow and thereby prevent acidification of the viral interior. Here we...
Preparation of a minimum PSII core complex from spinach is described, containing four Mn per reaction center (RC) and exhibiting high O2 evolving activity [approximately 4000 micromol of O2 (mg of chl)(-1) x h(-1)]. The complex consists of the CP47 and CP43 chlorophyll binding proteins, the RC D1/D2 pair, the cytochrome b559 subunits, and the Mn-stabilizing psbO (33 kDa) protein, all present in the same stoichiometric amounts found in the parent PSII membranes. Several small subunits are also present. The cyt b559 content is 1.0 per RC in core complexes and PSII membranes. The total chlorophyll content is 32 chl a and <1 chl b per RC, the lowest yet reported for any active PSII preparation. The core complex exhibits the characteristic EPR signals seen in the S2 state of higher plant PSII. A procedure for preparing low-temperature samples of very high optical quality is developed, allowing detailed optical studies in the S1 and S2 states of the system to be made. Optical absorption, CD, and MCD spectra reveal unprecedented detail, including a prominent, well-resolved feature at 683.5 nm (14630 cm(-1)) with a weaker partner at 187 cm(-1) to higher energy. On the basis of band intensity, CD, and MCD arguments, these features are identified as the exciton split components of P680 in an intact, active reaction center special pair. Comparisons are made with solubilized D1/D2/cyt b559 material and cyanobacterial PSII.
Low-temperature absorption and fluorescence spectra of fully active cores and membrane-bound PS II preparations are compared. Detailed temperature dependence of fluorescence spectra between 5 and 70 K are presented as well as 1.7-K fluorescence line-narrowed (FLN) spectra of cores, confirming that PS II emission is composite. Spectra are compared to those reported for LHCII, CP43, CP47 and D1/D2/cytit b559 subunits of PS II. A combination of subunit spectra cannot account for emission of active PS II. The complex temperature dependence of PS II fluorescence is interpretable by noting that excitation transfer from CP43 and CP47 to the reaction centre is slow, and strongly dependent on the precise energy at which a 'slow-transfer' pigment in CP43 or CP47 is located within its inhomogeneous distribution. PS II fluorescence arises from CP43 and CP47 'slow-transfer' states, convolved by this dependence. At higher temperatures, thermally activated excitation transfer to the PS II charge-separating system bypasses such bottlenecks. As the charge-separating state of active PS II absorbs at >700 nm, PS II emission in the 680-700 nm region is unlikely to arise from reaction centre pigments. PS II emission at physiological temperatures is discussed in terms of these results.
We review our recent low-temperature absorption, circular dichroism (CD), magnetic CD (MCD), fluorescence and laser-selective measurements of oxygen-evolving Photosystem II (PSII) core complexes and their constituent CP 4 3, CP 47 and D1/D2/cytb(559) sub-assemblies. Quantitative comparisons reveal that neither absorption nor fluorescence spectra of core complexes are simple additive combinations of the spectra of the sub-assemblies. The absorption spectrum of the D1/D2/cytb(559) component embedded within the core complex appears significantly better structured and red-shifted compared to that of the isolated sub-assembly. A characteristic MCD reduction or 'deficit' is a useful signature for the central chlorins in the reaction centre. We note a congruence of the MCD deficit spectra of the isolated D1/D2/cytb(559) sub-assemblies to their laser-induced transient bleaches associated with P 680. A comparison of spectra of core complexes prepared from different organisms helps distinguish features due to inner light-harvesting assemblies and the central reaction-centre chlorins. Electrochromic spectral shifts in core complexes that occur following low-temperature illumination of active core complexes arise from efficient charge separation and subsequent plastoquinone anion (Q(A)(-)) formation. Such measurements allow determinations of both charge-separation efficiencies and spectral characteristics of the primary acceptor, Pheo(D1). Efficient charge separation occurs with excitation wavelengths as long as 700 nm despite the illuminations being performed at 1.7 K and with an extremely low level of incident power density. A weak, homogeneously broadened, charge-separating state of PSII lies obscured beneath the CP 47 state centered at 690 nm. We present new data in the 690-760 nm region, clearly identifying a band extending to 730 nm. Active core complexes show remarkably strong persistent spectral hole-burning activity in spectral regions attributable to CP 43 and CP 47. Measurements of homogeneous hole-widths have established that, at low temperatures, excitation transfer from these inner light-harvesting assemblies to the reaction centre occurs with approximately 70-270 ps(-1) rates, when the quinone acceptor is reduced. The rate is slower for lower-energy sub-populations of an inhomogeneously broadened antenna (trap) pigment. The complex low-temperature fluorescence behaviour seen in PSII is explicable in terms of slow excitation transfer from traps to the weak low-energy charge-separating state and transfer to the more intense reaction-centre excitations near 685 nm. The nature and origin of the charge-separating state in oxygen-evolving PSII preparations is briefly discussed.
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