Reduction and protonation of the secondary quinone acceptor of Rhodobacter sphaeroides photosynthetic reaction center: kinetic model based on a comparison of wild-type chromatophores with mutants carrying Arg-->Ile substitution at sites 207 and 217 in the L-subunit.

After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone QA, the loosely bound ubiquinone Q(B) After two subsequent flashes of light, Q(B) is reduced to ubiquinol Q(B)H2, with a semiquinone anion Q-(B) formed as an intermediate after the first flash. We studied Q(B)H2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg-->Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 A away from Q(B), Arg-L217 is closer (9 A) and contacts the Q(B)-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated deltaG(AB), the free energy difference between the Q-(A)Q(B) and Q(A)Q-(B) states, and pK212, the apparent pK of Glu-L212, a residue that is only 4 A away from Q(B). As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q-(B) state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, pK212 increased by approximately 0.4 pH units in the L207RI mutant. The value of pK212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic parameters of the Q(B) turnover. The non-electrostatic, 'chemical' affinity of the QB site to protons proved to be as important for the attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established pK2J2 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the QB turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle.

Domain organization and flavin adenine dinucleotide-binding determinants in the aerotaxis signal transducer Aer of Escherichia coli.

Aerotactic responses in Escherichia coli are mediated by the membrane transducer Aer, a recently identified member of the superfamily of PAS domain proteins, which includes sensors of light, oxygen, and redox state. Initial studies of Aer suggested that it might use a flavin adenine dinucleotide (FAD) prosthetic group to monitor cellular redox changes. To test this idea, we purified lauryl maltoside-solubilized Aer protein by His-tag affinity chromatography and showed by high performance liquid chromatography, mass spectrometry, and absorbance spectroscopy that it bound FAD noncovalently. Polypeptide fragments spanning the N-terminal 290 residues of Aer, which contains the PAS motif, were able to bind FAD. Fusion of this portion of Aer to the flagellar signaling domain of Tsr, the serine chemoreceptor, yielded a functional aerotaxis transducer, demonstrating that the FAD-binding portion of Aer is sufficient for aerosensing. Aerotaxis-defective missense mutants identified two regions, in addition to the PAS domain, that play roles in FAD binding. Those regions flank a central hydrophobic segment needed to anchor Aer to the cytoplasmic membrane. They might contact the FAD ligand directly or stabilize the FAD-binding pocket. However, their lack of sequence conservation in Aer homologs of other bacteria suggests that they play less direct roles in FAD binding. One or both regions probably also play important roles in transmitting stimulus-induced conformational changes to the C-terminal flagellar signaling domain to trigger aerotactic behavioral responses.

A signal transducer for aerotaxis in Escherichia coli.

The newly discovered aer locus of Escherichia coli encodes a 506-residue protein with an N terminus that resembles the NifL aerosensor and a C terminus that resembles the flagellar signaling domain of methyl-accepting chemoreceptors. Deletion mutants lacking a functional Aer protein failed to congregate around air bubbles or follow oxygen gradients in soft agar plates. Membranes with overexpressed Aer protein also contained high levels of noncovalently associated flavin adenine dinucleotide (FAD). We propose that Aer is a flavoprotein that mediates positive aerotactic responses in E. coli. Aer may use its FAD prosthetic group as a cellular redox sensor to monitor environmental oxygen levels.

Mechanisms of oxygen taxis in bacteria.

Since 1881 when Englemann reported aerotaxis in bacteria, an understanding of the molecular nature of the signal transduction remains a daring goal for microbiologists. This short review discusses known facts and recent advances in the field including the discovery of the flavoprotein receptor which drives Escherichia coli towards oxygen. Possible mechanisms of oxygen sensing in various bacterial species are considered in connection with the existing, often fragmental, data on phototaxis, redox taxis and taxis repellent effect of the reactive oxygen species (ROS).

delta psi-mediated signalling in the bacteriorhodopsin-dependent photoresponse.

It has been shown previously that the proton-pumping activity of bacteriorhodopsin from Halobacterium salinarium can transmit an attractant signal to the bacterial flagella upon an increase in light intensity over a wide range of wavelengths. Here, we studied the effect of blue light on phototactic responses by the mutant strain Pho8l-B4, which lacks both sensory rhodopsins but has the ability to synthesize bacteriorhodopsin. Under conditions in which bacteriorhodopsin was largely accumulated as the M412 bacteriorhodopsin photocycle intermediate, halobacterial cells responded to blue light as a repellent. This response was pronounced when the membrane electric potential level was high in the presence of arginine, active oxygen consumption, or high-background long-wavelength light intensity but was inhibited by an uncoupler of oxidative phosphorylation (carbonyl cyanide 3-chlorophenylhydrazone) and was inverted in a background of low long-wavelength light intensity. The response to changes in the intensity of blue light under high background light was asymmetric, since removal of blue light did not produce an expected suppression of reversals. Addition of ammonium acetate, which is known to reduce the pH gradient changes across the membrane, did not inhibit the repellent effect of blue light, while the discharge of the membrane electric potential by tetraphenylphosphonium ions inhibited this sensory reaction. We conclude that the primary signal from bacteriorhodopsin to the sensory pathway involves changes in membrane potential.

Mechanism of photosensory adaptation in Halobacterium salinarium.

Phototaxis in Halobacterium salinarium is the result of an interplay of sensory rhodopsin excitation and adaptation to the stimulus background. Adaptation to orange light, received by sensory rhodopsin I was probed by measuring the behavioral response of cells to a step-like decrease in intensity. Cells were able to adapt to an intensity range of more than four orders of magnitude. The data were analysed on the basis of theoretical fluence rate response relationships calculated from the photocycle kinetics of the complex of sensory rhodopsin I with its transducer HtrI. Independent of the stimulus background, the cellular response was shown to be a function of the absolute number of photoreceptor complex molecules turned over by the light stimulus. Receptor deactivation was identified as the underlying mechanism of adaptation and was sufficient to account for the experimental results. We suggest that reversible methylation of the transducer protein HtrI provides the chemical mechanism of sensory adaptation in H. salinarium and also explains the different sensitivity of the cells to orange and UV light.

Flash-induced electrogenic reactions in the SA(L223) reaction center mutant in Rhodobacter sphaeroides chromatophores.

The charge transfer events in the SA(L223) reaction center mutant Rhodobacter sphaeroides chromatophores were investigated by direct electrometry. Besides the primary charge separation, the small stigmatellin-sensitive electrogenic reaction due to the electron transfer from the primary to the secondary quinone acceptor in the reaction center complex was observed after the first flash. The second flash-induced electrogenic phase of the secondary quinone protonation and subsequent electrogenic reactions of the cytochrome bc1 complex were much slower than those in chromatophores of the wild type. It is suggested that replacement of Ser-L223 by Ala impairs both specific proton-conducting pathways leading to the secondary quinone QB.

Bacteriorhodopsin is involved in halobacterial photoreception.

The bacterio-opsin gene was introduced into a "blind" Halobacterium salinarium mutant that (i) lacked all the four retinal proteins [bacteriorhodopsin (BR), halorhodopsin, and sensory rhodopsins (SRs) I and II] and the transducer protein for SRI and (ii) showed neither attractant response to long wavelength light nor repellent response to short wavelength light. The resulting transformed cells acquired the capability to sense light stimuli. The cells accumulated in a light spot, demonstrating the BR-mediated orientation in spatial light gradients. As in wild-type cells, a decrease in the intensity of long wavelength light caused a repellent response by inducing reversals of swimming direction, but, in contrast to wild-type cells, a decrease in the intensity of short wavelength light also repelled the cells. An increase in light intensity evoked an attractant response (i.e., a transient suppression of spontaneous reversals). Signal processing times and adaptation kinetics were similar to the SRI-mediated reactions. However, compared to SR-mediated photoresponses, higher light intensities were necessary to induce the BR-mediated responses. The light sensitivity of the transformant was increased by adding 1 mM cyanide and decreased by the addition of arginine, agents that respectively reduce and increase the light-independent generation of the electrochemical potential difference of H+ ions (delta mu H+). A decrease in irradiance to an intensity that was still high enough to saturate BR-initiated delta mu H+ changes failed to induce the repellent effect, but the addition of a protonophorous uncoupler sensitized the cell to these light stimuli. The BR D96N mutant (Asp-96 is replaced by Asn) with decreased proton pump activity showed strongly reduced BR-mediated responses. Azide, which increases this mutant's H+ pump efficiency, increased the photosensitivity of the mutant cells. Moreover, azide diminished (i) the membrane potential decreasing and (ii) repellent effects of blue light added to the orange background illumination in this mutant. We conclude that the BR-mediated photoreception is due to the light-dependent generation of delta mu H+. Our data are consistent with the assumption that the H. salinarium cell monitors the membrane energization level with a "protometer" system measuring total delta mu H+ changes or its electric potential difference component.

The proton pump bacteriorhodopsin is a photoreceptor for signal transduction in Halobacterium halobium.

Halobacterium halobium swims by rotating its polarly inserted flagellar bundle. The cells are attracted by green-to-orange light which they can use for photophosphorylation but flee damaging blue or ultraviolet light. It is generally believed that this kind of 'colour vision' is achieved by the combined action of two photoreceptor proteins, sensory rhodopsins-I and -II, that switch in the light the rotational sense of the bundle and in consequence the swimming direction of a cell. By expressing the bacteriorhodopsin gene in a photoreceptor-negative background we have now demonstrated the existence of a proton-motive force sensor (protometer) and the function of bacteriorhodopsin as an additional photoreceptor covering the high intensity range. When the bacteriorhodopsin-generated proton-motive force drops caused by a sudden decrease in light intensity, the cells respond by reversing their swimming direction. This response does not occur when the proton-motive force is saturated by respiration or fermentation.