Concerning the enigmatic cytochrome b-559 of oxygenic photosynthesis.

Although there is an extensive literature on the properties and possible electron transfer pathways of cytochrome b-559, which is a prominent subunit of the multi-subunit photosystem II complex which functions in oxygenic photosynthesis, there is presently no consensus on the function of b-559 in the photosynthetic electron transport chain. The inability in earlier times to define a redox-linked function of this cytochrome was, to a large extent, a consequence of an absence of biochemical and structure information to complement an extensive array of spectrophotometric studies of the cytochrome in situ. Based on the location of hetero-dimeric b-559 in the photosystem II reaction center complex, derived from crystal crystallographic structure analysis, and the absence of a necessary redox function for the cytochrome in PSII, it is proposed that the main function of cytochrome b-559 is linked to its role as a structure component in the PSII reaction center complex. This function resides in the association of b-559 through its heme histidine residues in the trans-membrane domains of the PsbE and PsbF subunits of the PSII reaction center. These subunits, along with PsbJ, are inferred, from the analysis of structure, to define the intra-membrane portal in the PSII reaction center for plastoquinol (PQH2) export which, through the PSII complex, provides the redox link to the cytochrome b6f complex in the electron transfer chain.

On mechanisms of colicin import: the outer membrane quandary.

Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (Kd ≈ 10-9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.

Pathways of Transmembrane Electron Transfer in Cytochrome bc Complexes: Dielectric Heterogeneity and Interheme Coulombic Interactions.

The intramembrane cytochrome bc1 complex of the photosynthetic bacterium Rhodobacter capsulatus and the cytochrome b6f complex, which functions in oxygenic photosynthesis, utilize two pairs of b-hemes in a symmetric dimer to accomplish proton-coupled electron transfer. The transmembrane electron transfer pathway in each complex was identified through the novel use of heme Soret band excitonic circular dichroism (CD) spectra, for which the responsible heme-heme interactions were determined from crystal structures. Kinetics of heme reduction and CD amplitude change were measured simultaneously. For bc1, in which the redox potentials of the transmembrane heme pair are separated by 160 mV, heme reduction occurs preferentially to the higher-potential intermonomer heme pair on the electronegative (n) side of the complex. This contrasts with the b6f complex, where the redox potential difference between transmembrane intramonomer p- and n-side hemes is substantially smaller and the n-p pair is preferentially reduced. Limits on the dielectric constant between intramonomer hemes were calculated from the interheme distance and the redox potential difference, ΔEm. The difference in preferred reduction pathway is a consequence of the larger ΔEm between n- and p-side hemes in bc1, which favors the reduction of n-side hemes and cannot be offset by decreased repulsive Coulombic interactions between intramonomer hemes.

[Ortho/para spin-isomers of H2O molecules as a factor responsible for formation of two structural motifs in water].

According to the last results obtained by small-angle X-ray scattering and X-ray spectroscopy it was suggested that water within the nanometer scale represents a fluctuating mixture of clusters with tetrahedral structure and a subphase with partially broken hydrogen bonds whereas the nuclear configuration of the H20 molecule corresponds to single tetrahedral coordination. The basic reason of such structural partition is not clear until now. Here we show that it can be associated with the existence of two nuclear H2O spin-isomers which have different probability to be in one or another subphase. The para-molecule can transfer an excess of its rotational energy to the environment up to the complete stopping of rotation because its rotational quantum number J = 0 in the basic state. This property is favorable for the formation of clusters with closed H-bonds. Ortho-molecules with odd-numbered J states lack for this property and thus should be predominantly present in the surrounding with distorted bonds.

[Structural compatibility of hydration shells as a criterion of mutual recognition of reacting biomolecules].

Before a biochemical reaction begins to occur reagents need to recognize each other. To explain the recognition mechanism the "key-lock" hypothesis has been proposed at the end of the nineteenth century. Now it should be reconsidered since in the overwhelming majority of cases surface landscapes of the interacting molecules -- individually and in the complex -- do not coincide. The subsequent modifications of this hypothesis, for instance, the ligand-induced adjustment, were unable to predict a degree of the partner affinity and alleviate decision-making in essential task as to the search for the new drug forms. Here we offer a concept according to which the approaching reagents recognize each other by their hydration shells. Such an approach is based on the existence of universal structural blocks in water skeleton of biomolecules. The mutual recognition occurs when hydration shells of reagents are structurally compatible. This statement is demonstrated by the example of the simplest biochemical reaction.

[A structural criterion for the biological compatibility of materials].

The principles of searching and selection of the polymeric materials compatible with biological tissues are offered, based on structural features of the hydration shells of biopolymers.

[Water: nanostructure and fluctuations].

Recently a model of local organization of water was experimentally justified, in which tetrahedrally coordinated water clusters of 1-2 nanometers arise and disappear in liquid composed of H2O molecules with partially broken hydrogen bonds [1, 2]. Here we show that the clusters can oscillate between two structural forms, of which one is common hexagonal ice Ih whereas another is formed of the modules participating in formation of hydration shells of biomolecules. It is suggested that such self-oscillations are responsible for observable fluctuations of various physical and chemical parameters of water.

Membrane proteins in four acts: function precedes structure determination.

Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) ß-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to OmpF established the role of OmpF as an import channel for colicin nuclease cytotoxins. (IV) α-Synuclein, associated with the etiology of Parkinson's Disease, is an example of a protein, which is soluble and disordered in solution, but which can assume an ordered predominantly α-helical conformation upon binding to membranes. When subjected in its membrane-bound form to a trans-membrane electrical potential, α-synuclein can form voltage-gated ion channels. Summary of methods to assay functions/activities: (i) sensitive spectrophotometric assay to measure electron transfer activities; (ii) hydrophobic chromatography to deplete lipids, allowing reconstitution with specific lipids for studies on lipid-protein interactions; (iii) microbiological screen to assay high affinity binding of colicin receptor domains to Escherichia coli outer membrane receptors; (iv) electrophysiology/channel analysis (a) to select channel-occluding ligands for co-crystallization with ion channels of OmpF, and (b) to provide a unique description of voltage-gated ion channels of α-synuclein.

[Possible fundamental unity of magnetobiological "resonances"].

Organisms exposed to a combination of weak, parallel directed static and alternate magnetic fields show a distinct response when the frequency of the alternate component is formally equal to the cyclotron frequencies for Ca2+ or other biologically important ions. It is impossible to explain the observable phenomenon through a magnetoinduced drift of the ions, as the Lorentz's force is too small to change ionic movements. In similar conditions, a resonance-like response arises when the alternate field is tuned to the Larmor frequency for nuclear-spin magnetic moments. The mechanism of these phenomena is also still unclear. In the report, the arguments are presented to treat both types of effects in a single context for which the existence of ion magnetic dipoles is postulated.

Lipid-mediated inactivation of colicin E1 channels by calcium ions.

Based on the model of a toroidal protein-lipid pore, the effect of calcium ions on colicin E1 channel was predicted. In electrophysiological experiments Ca2+ suppressed the activity of colicin E1 channels in membranes formed of diphytanoylphosphatidylglycerol, whereas no desorption of the protein occurred from the membrane surface. The effect of Ca2+ was not observed on membranes formed of diphytanoylphosphatidylcholine. Single-channel measurements revealed that Ca2+-induced reduction of the colicin-induced current across the negatively charged membrane was due to a decrease in the number of open colicin channels and not changes in their properties. In line with the toroidal model, the effect of Ca2+ on the colicin E1 channel-forming activity is explained by alteration of the membrane lipid curvature caused by electrostatic interaction of Ca2+ with negatively charged lipid head groups.

Chemical and photochemical modification of colicin E1 and gramicidin A in bilayer lipid membranes.

Chemical modification and photodynamic treatment of the colicin E1 channel-forming domain (P178) in vesicular and planar bilayer lipid membranes (BLMs) was used to elucidate the role of tryptophan residues in colicin E1 channel activity. Modification of colicin tryptophan residues by N-bromosuccinimide (NBS), as judged by the loss of tryptophan fluorescence, resulted in complete suppression of wild-type P178 channel activity in BLMs formed from fully saturated (diphytanoyl) phospholipids, both at the macroscopic-current and single-channel levels. The similar effect on both the tryptophan fluorescence and the electric current across BLM was observed also after NBS treatment of gramicidin channels. Of the single-tryptophan P178 mutants studied, W460 showed the highest sensitivity to NBS treatment, pointing to the importance of the water-exposed Trp460 in colicin channel activity. In line with previous work, the photodynamic treatment (illumination with visible light in the presence of a photosensitizer) led to suppression of P178 channel activity in diphytanoyl-phospholipid membranes concomitant with the damage to tryptophan residues detected here by a decrease in tryptophan fluorescence. The present work revealed novel effects: activation of P178 channels as a result of both NBS and photodynamic treatments was observed with BLMs formed from unsaturated (dioleoyl) phospholipids. These phenomena are ascribed to the effect of oxidative modification of double-bond-containing lipids on P178 channel formation. The pronounced stimulation of the colicin-mediated ionic current observed after both pretreatment with NBS and sensitized photomodification of the BLMs support the idea that distortion of membrane structure can facilitate channel formation.

Insertion intermediates of pore-forming colicins in membrane two-dimensional space.

The formation of integral membrane voltage-gated ion channels by the initially soluble C-terminal channel polypeptide (CP) of the pore-forming colicins is a fruitful area for studies on membrane protein import. The dependence of CP import on specific membrane parameters can be better understood using liposomes and planar membranes of defined lipid composition. The membrane surface and interfacial layer provide special conditions for the transition of a pore-forming colicin from the soluble to the integral membrane state. The colicin E1 CP is arranged in the membrane interfacial layer as a conformationally mobile helical array that is extended far more in the two dimensions parallel to the membrane surface than in the third dimension perpendicular to it. The alpha-helical content of CP(E1) increases by approximately 30% upon binding to the membrane. The sequence of kinetically distinguishable events in the CP(E1)-membrane interaction is binding, unfolding to a subtended area of 4200 A(2), helix extension, and insertion, the last three events overlapping in their time course ( approximately 10 s(-1)). The extension into two dimensions and the interaction with the membrane surface may explain the reversible denaturation and refolding of secondary structure that occurs after boiling of the CP-membrane complex. Although DSC showed the presence of helix-helix interactions in the membrane-bound state, the change in secondary structure and the extended surface area argue against a molten-globule intermediate in the CP-membrane interaction. However, the surface-bound state is mobile, as surface conformational mobility is a necessary prerequisite for insertion of CP trans-membrane helices into the bilayer. The requirement for this surface protein mobility, described by "thermal melting" FRET experiments, may provide the explanation for the precipitous decrease in the voltage-gated CP channel formation at high values of surface potential of planar bilayer membranes. Thus, the membrane interfacial layer, with the CP backbone situated near the acyl chain carbonyls, provides a favorable environment for the structure changes necessary for the transition from the soluble to the membrane-inserted state.

[Influence of microwave and laser radiation on survivability of organisms]. / Vliianie mikrovolnomogo i lazernogo izlucheniia na zhiznesposobnosts' organizmov.

Depending on energy, spatial-temporal, modulation and other characteristics of an electromagnetic field, properties of a subject, exposure settings and possible interaction of other attendant factors microwaves may have either stabilizing, wholesome and even therapeutic or negative (damaging) effects on biological and ecological objects and systems. Therefore, there are two interrelated problems to be addressed. One is electromagnetic safety of and health provisions for humans exposed to EMF and EMR and the other, effective utilization of microwave EMF and EMR for biomedical and other purposes associated with enhancement of viability of organisms. The light-oxygen effect of laser radiation is gaining footing in therapy where it is used to activate or destroy biological systems by optical radiation at a specified light dose. Thus, low-intensity laser radiation can be used to improve viability and high-intensity laser radiation, to treat cancers.

Tryptophan-dependent sensitized photoinactivation of colicin E1 channels in bilayer lipid membranes.

The bacterial toxin colicin E1 is known to induce voltage-gated currents across a planar bilayer lipid membrane. In the present study, it is shown that the colicin-induced current decreased substantially upon illumination of the membrane in the presence of the photosensitizer, aluminum phthalocyanine. This effect was almost completely abolished by the singlet oxygen quencher, sodium azide. Using single tryptophan mutants of colicin E1, Trp495 was identified as the amino acid residue responsible for the sensitized photodamage of the colicin channel activity. Thus, the distinct participation of a specific amino acid residue in the sensitized photoinactivation of a defined protein function was demonstrated. It is suggested that Trp495 is critical for the translocation and/or anchoring of the colicin channel domain in the membrane.

Folded state of the integral membrane colicin E1 immunity protein in solvents of mixed polarity.

The colicin E1 immunity protein (ImmE1), a 13.2-kDa hydrophobic integral membrane protein localized in the Escherichia coli cytoplasmic membrane, protects the cell from the lethal, channel-forming activity of the bacteriocin, colicin E1. Utilizing its solubility in organic solvents, ImmE1 was purified by 1-butanol extraction of isolated membranes, followed by gel filtration and ion-exchange chromatography in a chloroform/methanol/H(2)O (4:4:1) solvent system. Circular dichroism analysis indicated that the alpha-helical content of ImmE1 is approximately 80% in 1-butanol or 2,2,2-trifluoroethanol, consistent with a previous membrane-folding model with three extended hydrophobic transmembrane helical domains, H1-H3. Each of these extended hydrophobic domains contains a centrally located single Cys residue that could be used as a probe of protein structure. The presence of tertiary structure of purified ImmE1 in a solvent of mixed polarity, chloroform/methanol/H(2)O (4:4:1) was demonstrated by (i) the constraints on Tyr residues shown by the amplitude of near-UV circular dichroism spectra in the wavelength interval, 270-285 nm; (ii) the correlation between the near-UV Tyr CD spectrum of single and double Cys-to-X mutants of the Imm protein and their in vivo activity; (iii) the upfield shift of methyl groups in a 1D NMR spectrum, a 2D- HSQC NMR spectrum of ImmE1 in the mixed polarity solvent mixture, and a broadening and disappearance of the indole (1)H proton resonance from Trp94 in H3 by a spin label attached to Cys16 in the H2 hydrophobic domain; (iv) near-UV circular dichroism spectra with a prominent ellipticity band centered at 290 nm from a single Trp inserted into the extended hydrophobic domains. It was concluded that the colicin E1 immunity protein adopts a folded conformation in chloroform/methanol/H(2)O (4:4:1) that is stabilized by helix-helix interactions. Analysis of the probable membrane folding topology indicated that several Tyr residues in the bilayer region of the three transmembrane helices could contribute to the near-UV CD spectrum through helix-helix interactions.

Structural stability and domain organization of colicin E1.

Thermodynamic properties, stability, and structure of the toxin-like molecule colicin E1 were analyzed by differential scanning calorimetry and circular dichroism to determine the number of structurally independent domains, and the interdomain interactions necessary for colicin import into the Escherichia coli cell. Analysis of denaturation profiles of the 522 residue colicin E1, together with fragments of 342 and 178 residues that contain subsets of the domains, showed three stable cooperative blocks that differ in thermal stability and correspond to three major functional domains of the colicin: (i) the COOH-terminal channel-forming (C) domain with the highest thermal stability; (ii) the BtuB receptor binding (R) domain; and (iii) the N-terminal translocation (T) domain that has the smallest stabilization enthalpy and thermal stability. Interdomain interactions were described in which T-R interactions stabilize R, and T-C and R-C interactions stabilize R and T, but destabilize C. The R and T domains behaved in a similar way as a function of pH and ionic strength. Interacting extended helices of the R domain, possibly a coiled-coil, were implied by: (i) the very high (>90%) alpha-helical content of the R domain, (ii) cooperative decreases in alpha-helical content near the T(tr) of thermal denaturation of the R domain; (iii) a large denaturation enthalpy, implying extensive H-bond and van der Waals interactions. The R domain was inferred, from the extended network of interacting helices, large DeltaH, and steep temperature dependence of its stabilization energy to have a dominant role in determining the conformation of other domains. It is proposed that cellular import starts with the R domain binding to the BtuB receptor, followed by unfolding of the R domain coiled-coil and thereby of the T domain, which then interacts with the TolC receptor-translocator.

Unfolding pathway of the colicin E1 channel protein on a membrane surface.

The channel-forming domain of colicin E1 is composed of a soluble helical bundle which, upon membrane binding, unfolds to form an extended, two-dimensional helical net in the membrane interfacial layer. To characterize the pathway of unfolding of the protein and the structure of the surface-bound intermediate, the time-course of intra-protein distance changes and unfolding on a millisecond time-scale were determined from the kinetics of changes in the efficiency of fluorescence resonance energy transfer, and of the donor-acceptor overlap integral, between each of six individual tryptophan residues and a Cys-conjugated energy transfer acceptor (C509-AEDANS). Comparison of the rate constants revealed the following order of events associated with unfolding of the protein at the membrane surface: (A) movement of the hydrophobic core helices VIII-IX, coincident with a small change in Trp-Cys509 distances of the outer helices; (B) unfolding of surface helices in the helical bundle in the order: helix I, helices III, IV, VI, VII, and helix V; (C) a slow (time-scale, seconds) condensation of the surface-bound helices. The rate of protein unfolding events increased with increasing anionic lipid content. Unfolding did not occur below the lipid thermal phase transition, indicating that unfolding requires mobility in the interfacial layer. The structure of the two-dimensional membrane-bound intermediate in the steady-state was inferred to consist of a quasi-circular arrangement of eight helices embedded in the membrane interfacial layer and anchored by the hydrophobic helical hairpin. The pathway of unfolding of the colicin channel at the membrane surface, catalyzed by electrostatic and hydrophobic forces, is the first described for a membrane-active protein. It is proposed that the pathway and principles described for the colicin protein are relevant to membrane protein import.

Kinetic description of structural changes linked to membrane import of the colicin E1 channel protein.

Upon binding to membranes, the 178-residue colicin E1 C-terminal channel protein forms a steady-state closed-channel intermediate that is a flexible extended two-dimensional helical array [Zakharov et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4282-4287]. Analysis of the kinetics of binding-insertion to liposome membranes of the channel protein, P178, and of changes of spectral parameters associated with structure transitions allowed a correlation of the sequence of tertiary and secondary structure changes with binding-insertion. Binding and insertion were distinguished by use of lipids modified with quenchers of Trp fluorescence attached to lipid headgroups or acyl chains. Secondary and tertiary structure changes were inferred, respectively, from changes in far-UV circular dichroism and relative changes of interresidue distances by fluorescence resonance energy transfer (FRET). "Single Trp" mutants were used in FRET analysis, with the background Tyr contribution determined through use of a "zero Trp" mutant. The sequence of distinguishable events and the pseudo-first-order rate constants under "standard" conditions (large unilamellar vesicles, pH 4.0, I = 0.1 M) was binding (30 +/- 5 s(-)(1)) --> unfolding (12.6 +/- 0.5 s(-)(1)) --> helix elongation (9.0 +/- 1.0 s(-)(1)) --> insertion (6. 6 +/- 0.5 s(-)(1)). Thus, helix elongation on the surface of the membrane can occur after unfolding and does not require insertion. Binding-insertion and structural transitions of P178 occur significantly faster with small unilamellar vesicles. The relevance to general mechanisms of protein import of the structural changes associated with import of the colicin channel is discussed.

Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array.

Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the alpha-helical content increases from 60-64% to 80-90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 A2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled alpha-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.

Calcium binding to the subunit c of E. coli ATP-synthase and possible functional implications in energy coupling.

The 8-kDa subunit c of the E. coli F0 ATP-synthase proton channel was tested for Ca++ binding activity using a 45Ca++ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds 45Ca++ strongly with Ca++ binding properties very similar to those of the 8-kDa CF0 subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca++ binding capacity, shown by loss of Ca++ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca++ binding, shown by Ca++ binding being retained in two E. coli mutants, Asp61-->Asn and Asp61-->Gly. The Ca++ binding is pH dependent in both the E. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca++ binding affinity to its F0 binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca++ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca++ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80-100 nM was obtained using an EGTA-Ca++ buffer system to control free Ca++ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca++ bound to the periplasimic side of the E. coli F0 proton channel can block H+ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca++ binding site is on the lumen side of the thylakoid, where Ca+2 binding can modulate acid-base jump ATP formation. The Ca+2 binding to the F0 and CF0 complexes is consistent with a pH-dependent gating mechanism for control of H+ ion flux across the opening of the H+ channel.