Light absorbed by

bacteriorhodopsin can drive the creation of a ∆ p

Certain archaea, namely, the extremely halo- philic archaea, have evolved a way to produce a ∆p by using light energy directly (i.e., without the intervention of oxidation–reduction reac- tions and without chlorophyll).65–67 _{See note 68}

for a more complete discussion of the extreme halophiles. (It has been reported that under the proper conditions, these microorganisms can be grown photoheterotrophically, i.e., on organic carbon with light as a source of energy, but such conclusions have been questioned.69,70₎

Halophilic archaea are heterotrophic organisms that carry out an ordinary aerobic respiration, creating a ∆p driven by oxidation– reduction reactions during electron transport. (See note 71.) The ∆p is used to drive ATP synthesis via a membrane ATP synthase. (See Sections 4.7.1 and 4.6.2.) However, conditions for respiration are not always optimal and, in the presence of light and low oxygen levels, the halophiles adapt by making photopigments (rhodopsins), one of which (bacteriorhodopsin) functions as a proton pump that is energized directly by light energy. (See note 72.) Whereas photosynthetic electron fl ow is an example of an indirect transformation of light energy into an electrochemical potential (via redox reac- tions), bacteriorhodopsin illustrates the direct transformation of light energy into an electro- chemical potential. We will fi rst consider data

that demonstrate the light-dependent pumping of protons. We will then describe the proton pump, the photocycle, and a model for the mech- anism of pumping protons. Bacteriorhodopsin is examined in detail because it is the best char- acterized ion pump.

Evidence that halophiles can use light energy to drive a proton pump

Figure 4.18 models the results of subjecting a suspension of Halobacterium halobium to light of different intensities and for different periods of time, during which the pH of the external medium was monitored. As illustrated in Fig. 4.18A, the light produced an effl ux of protons from the cell. Higher light intensities produced a greater rate of proton effl ux (compare I_c to I_a.) In Fig. 4.18B, the rate of proton effl ux, in nanograms per second, as determined from the slopes in Fig. 4.18A, is plotted as a func- tion of the light intensity in nanoeinsteins per second (an einstein is equal to a “mole” of pho- tons). From data such as these, a quantum yield (i.e., protons ejected per photon absorbed) was calculated to be 0.52 proton per photon absorbed.73 _{The reported values for the maxi-}

mum quantum yield for proton effl ux is a little higher (0.6–0.7 proton/photon absorbed). The quantum yield for the photocycle (i.e., the frac- tion of bacteriorhodopsin molecules absorbing light that undergoes the photocycle described in the next subsection) is 0.64 ± 0.04.74,75 _These

values suggest that one proton is pumped per photocycle.

Bacteriorhodopsin is the proton pump

Built into the cell membrane of the halophilic archaebacteria is a pigment protein called bac- teriorhodopsin, which is a pump responsible for the light-driven electrogenic effl ux of pro- tons. It consists of one large polypeptide (248 amino acids, 26,486 Da) folded into seven α helices that form a transmembrane channel (Fig. 4.19). (See Ref. 76 for a review.) Located in the middle of the channel, and attached to the bacteriorhodopsin, is a pigment called reti- nal (a C₂₀ carotenoid), which is attached via a Schiff base to a lysine residue on the protein. (Note 77 tells what a Schiff base is.) When the retinal absorbs light, the bacteriorhodopsin remarkably translocates protons out of the cell, and a ∆p is created.

Fig. 4.18 Proton pumping by bacteriorhodopsin.

Expected results if Halobacterium cells were illu- minated by light. To measure proton outfl ow accu- rately, proton infl ow through the ATP synthase must be blocked either with uncouplers or nigericin, which collapse the proton electrochemical potential, or with an ATP synthase inhibitor such as DCCD. The extruded protons can be quantitated with a pH meter. (A) Proton effl ux measured as a function of time at increasing light intensities I_a < I_b < I_c, expressed as nanoeinsteins per second. The slope of each line is the rate of proton effl ux. (B) The rate of proton effl ux is plotted as a function of the light intensity. From these data one can calculate the quantum yield (i.e., the number of protons extruded per photon absorbed).

Source: Adapted from data by Bogomolni, R. A.,

R. A. Baker, R. H. Lozier, and W. Stoeckenius. 1980. Action spectrum and quantum effi ciency for proton pumping in Halobacterium halobium. Biochemistry

19:2152–2159.

Th e photocycle and a model for proton pumping

The photoevents occurring in halophiles can be followed spectroscopically because when bacteriorhodopsin (bR) absorbs light, it loses its absorption peak at 568 nm (bleaches) and is converted in the dark to a series of pigments that have absorption peaks at different wavelengths. This sequence of events is called the photocycle. Also, site-specifi c mutagenesis of bacteriorho- dopsin is being used to identify the amino acid side chains that transfer protons across the

membrane.78–81 _{The photocycle is shown in Fig.}

4.20. The retinal is attached via a Schiff base to the ε amino group of lysine-216 (K₂₁₆). Before absorbing light, the retinal is protonated at the Schiff base and exists in the all-trans (13-trans) confi guration (bR₅₆₈). The subscript refers to the absorption maximum, in nanometers. Upon absorbing a photon of light, the retinal isomerizes to the 13-cis form (K₆₂₅). All subse- quent steps do not require light and represent the de-energization of the bacteriorhodopsin via a series of intermediates that have absor- bance maxima different from those of the origi- nal unexcited molecule. The transitions are very fast and occur in the nanosecond and mil- lisecond ranges. These intermediates are, in the order of their appearance, K, L, M, N, and O.

In converting from L to M, the Schiff base loses its proton to aspartate-85 but regains a proton from aspartate-96 in going from M to

Fig. 4.19 Diagram of bacteriorhodopsin. The seven

helices, shown as rods, form a central channel; the retinal is attached to a lysine residue on helix G.

Source: Henderson, R., J. M. Baldwin, T. A. Ceska,

F. Zemlin, E. Beckmann, and K. H. Downing. 1990. Model for the structure of bacteriorhodopsin based on high-resolution electron cryomicroscopy. J. Mol.

Biol. 213:899–929.

N. Aspartate-96 acquires a proton from the cytoplasm in going from N to O. Thus, a proton has moved from the cytoplasmic side through aspartate-96 to the Schiff base to aspartate-85. From aspartate-85, the proton moves to the outside membrane surface. (See note 82.)

Precisely how the proton travels through the bacteriorhodopsin channel from the cytoplas- mic side to the Schiff base in the center of the channel, and from there to the external sur- face of the membrane, is not known. Probably the proton is passed from one amino acid side group that can be reversibly protonated to another. Site-specifi c mutagenesis experi- ments have suggested that two of these amino acids are aspartate-85 and aspartate-96. The protonatable residues would extend along the protein from the cytoplasmic surface to the out- side. In the case of bacteriorhodopsin, where the three-dimensional structure is known, the protonatable residues are oriented toward the center of the channel. Any bound water might also participate in proton translocation. For example, there might be a chain of water and hydrogen-bonded protons connecting protona- table groups. Since the Schiff base gives up its proton to the extracellular side of the channel (transition L to M) and becomes protonated with a proton from the cytoplasm (transition M to N), it has been assumed that there is a switch that reorients the Schiff base, causing it to face the cytoplasmic and extracellular sides of the channel alternately. Logically, the switch would be at M. The mechanism for the switch is unknown, although a conformational change in the bacteriorhodopsin has been suggested.80

It is not obviously correlated with the retinal isomerizations because the retinal is in the cis confi guration throughout most of the photocy- cle, including the protonation and deprotona- tion of the Schiff base. The transfer of protons along protonatable groups has been suggested for several proton pumps besides bacteriorho- dopsin, including cytochrome oxidase and the proton-translocating ATP synthase.83

4.9 Halorhodopsin, a Light-Driven