Received March 14, 2012
The artificial pigment 4-ketobacteriorhodopsin is an interesting analog of bacteriorhodopsin. Arguments concerning the scheme of the photocycle of 4-ketobacteriorhodopsin are discussed.
KEY WORDS: chromophore, retinal analogs, 13-cis-cycle, M-intermediate, M419, K570
Abbreviations: BR, bacteriorhodopsin; PM, purple membrane; Z-BR and E-BR, BR with retinal chromophore in 13-cis- and all-trans-configurations, respectively; λmax, maximum of the major band in the absorption spectrum of the chromophore; the lower index of an intermediate of the cycle (e.g. M412, K570) is its differential maximum.
Bacteriorhodopsin (BR) is a light-dependent generator of Δµ–H+ [1-5]. It is isolated from the bacterium Halobacterium salinarum (halobium) as purple membranes (PM). The chromophore of the pigment consists of the Schiff base of retinal and the ε-amino group of a lysine residue of the protein. The transfer of H+ occurs during a cyclic transformation of the pigment (the photocycle, see table). Chromophore modification is an approach to the study such pigments. The 4-ketoanalog combines high efficiency and a significant deceleration of the photocycle, and this is of importance for both scientific and applied problems [12-15]. Unfortunately, the unsuccessful scheme of the 4-ketoBR photocycle in a series of publications (table, item 7; [9-11]) leads to delay in discussion of new experimental data. The purpose of the present work is to show apparent errors in those publications.
Features of BR and its 4-ketoanalog
* For BR data are presented for PM, preparations of 4-ketoBR are membranes resulting on the interaction of apo-membranes and 4-ketoretinal.
** Above 0ºC and below the temperature of the first signs of thermal denaturation of the preparation (usually below 30ºC).
A group of biophysicists has proposed in 1991 a scheme of 4-ketoBR functioning , and the authors defended it later [10, 11], whereas a group from Pushchino supported it also in 2009 in interviews. These authors consider three peaks in the spectrum of M-intermediate of 4-ketoBR detected at –180ºC (table, item 6) to be a specific feature of 4-ketoBR and conclude that these peaks indicate three different M-containing cycles (table, item 7), whereas the 13Z-form enters one of the cycles and the E-conformer is a precursor of two other cycles. The authors of [9-11] include the recorded K570 into all three cycles of the scheme, do not notice the L-intermediate, doubt its presence in 4-ketoBR, and indicate a branch of cycle “shortening” from M to the initial state. However, there is no doubt that on prolonged illumination a rapid return is recorded considering that the photosensitivity of virtually all intermediates has been described long before [5, 16]; in particular, such transition (the so-called “blue inhibition”) is a well-studied phenomenon [16, 17]. Moreover, the photosensitivity of M in the cycle of the analog is not sufficient to indicate the branching M → (analog) BRin steady-state in the dark.
The vibronic structure of the M-intermediate of BR containing the natural retinal in low-temperature spectroscopy is also known [5-7] (table, item 3), but it does not suggest parallel cycles. Moreover, such ideas about the functioning mechanisms became out of date after publication of , which established the reversibility of the majority (or all) stages of the BR cycle. Maxima of low temperature peaks of BR and 4-ketoBR are rather close (table, items 3 and 6), and even more peaks have been found in BR (possibly because of increase resolution).
In works [9, 10] the accumulation of a photostationary long-lived M440 is an additional confirmation of the scheme, but phototransformations of intermediates are diverse , and most long-lived product will be accumulated, independent of its relation with a single turnover of the cycle.
As differentiated from the proton transfer cycle of E-BR, the Z-cycle under usual conditions has neither an M-intermediate nor H+ transfer, and the life of K-like intermediates is longer [1, 18, 19]. It was shown by Kaulen’s group [20-22] that at high pH the M-intermediate and proton transfer appear in the Z-cycle, but the decay of K accelerates to times typical for the E-cycle. Thus, depending on conditions, BR and its analogs either have in their cycles an M-like intermediate or specific long-lived long-wavelength intermediates [21-24]. We compared the kinetics of spectral transformations of individual forms of E- and Z-4-ketoBR and isomerized preparations containing a mixture of these forms , and we found that the E-cycle contains no long-wavelength intermediates, and all signals recorded in this region on uncontrolled preparations are a summary of K-like long-lived intermediates of the Z-cycle and a decrease in the optical density in the main band of the E-cycle specific absorption. Therefore, the assignment of M395 of 4-ketoBR to the Z-cycle under conditions discussed in [9, 10] (table, item 7) and K570 to all cycles of the E- and Z-forms of the analog seems to be unreasonable. An unclear situation with L is caused by a high amplitude of K570 which is specific just for the Z-cycle (a similar case has been analyzed in detail for phenyl analogs of BR ). We have recorded similar long-wavelength kinetics in the cycles of individual Z-forms of 4-ketoBR  and of other analogs [23-26], but no such kinetics have been recorded in the cycle of the individual E-form of 4-ketoBR . The comparison of λmax (table, items 4, 5, and 7) shows that in preparations of the authors of the scheme of the cycle [9-11] Z-4-ketoBR is prevalent (we have also studied similar mixed preparations [8, 12, 14]). The maximum of the differential spectrum of the E-form K-intermediate is further to the red than that of the Z-form [5, 18]. Therefore, it is reasonable to assign K570 only to the Z-cycle and exclude it from the scheme of the E-4-ketoBR cycle.
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