Dietrich Kuschmitz

Kinetic interaction between aromatic residues and the retinal chromophore of bacteriorhodopsin during the photocycle

In an earlier communication [ I] a reaction of the protein fluorescence following 570 nm light activation of bacteriorhodopsin (BR) was described, indicating the participation of the aromatic amino acid residues in the mechanism of its photoand protoncycle and a conformational change. Recently confirmation and extension of these data were presented suggesting a deprotonation and reprotonation of tyrosine and possibly tryptophane during the photocycle [2,3]. This notion is supported by the elucidation of the amino acid analysis of BR [4], the pH-sensitivity of its aromatic spectrum [S] as well as the results of chemical substitution experiments of tyrosine residues [6]. Here we report on the transient and steady state difference spectra of the aromatic amino acid residues, their reaction velocity constants, their approximate stoichiometry during a photocycle as well as on their interactions and time correlation with photocycle intermediates.

Chaotic dynamics in yeast glycolysis under periodic substrate input flux

The numerical analysis for a glycolytic model containing the enzymes phosphofructokinase and pyruvate kinase reveals different types of entrainment, as well as chaotic response under sinusoidal substrate input. Entrainment with response periods 1, 2, 3, 5 and 7‐times the input flux period and aperiodic behaviour is verified by measurements of NADH fluorescence in extracts of Saccharomyces cerevisiae in the theoretically predicted range. The stroboscopic transfer function obtained from the aperiodic signal admits period 3, implying chaos according to the Li‐Yorke theorem.

The photochemical reaction of the 412 nm chromophore of bacteriorhodopsin

The purple membrane of Halobacterium halobium converts light energy into electrochemical energy [ 1,2] . Light absorbed by Bacteriorhodopsin (BR 570) induces a reaction cycle which consists of two main parts: First, BR 570 is transformed to a relatively stable product absorbing at 412 nm (BR 4 12) under proton release into the medium, in the second part of the cycle BR 570 is regenerated under proton uptake [2-51. While in the first reaction sequence several short-life intermediates could be identified [4,6-81, the mechanism of the regeneration process is not understood. Some time ago, we observed the acceleration of the proton uptake of bleached Bacteriorhodopsin by illumination with blue light [2,9] , an interaction which proved to be useful for the study of the regeneration process. Here, we report that this light activated regeneration of Bacteriorhodopsin BR 570 fulfills the criteria of a photochemical reaction and that the regeneration kinetics can be described by two exponentials, which involve two different forms of the BR 412 chromophore, as well as a component in addition to the BR 570 chromophore, preliminary results of which have been reported elsewhere [9,10].

Branching reactions in the photocycle of bacteriorhodopsin

The purple membrane of Halobacterium halobium acts as a light-driven proton pump [ 11. It contains a single protein to which a retinal is bound via a protonated Schiff-base [2,3]. This protein-retinal complex, bacteriorhodopsin (BR), undergoes a reaction cycle involving several intermediates after light absorption. The photocycle can be described by the following scheme showing the time sequence of intermediates of BR in the purple membrane as defined in [4], the numbers giving their approximate wavelength maxima [4-61:

Trans—cis isomerisation of the retinal chromophore of bacteriorhodopsin during the photocycle

Since the ,discovery of the reversible photocycle of the retinal chromophore in bacteriorhodopsin (bR) the question was asked whether the retinal chromophore undergoes an isomerisation reaction during the photocycle such as observed in rhodopsin [ 1,2]. Indeed, the existence of all-Pans and 13-cis retinal isomers in bR, held in the dark (dark-adapted bR) [3-S] and the isomerisation of the 13-cis isomer into 100% all-trans isomer of bR by weak illumination (light adaptation) [4,5] was recognized. In [6] 13& retinal was extracted from bR kept in the M-412 state at low temperatures in the presence of guanidinium and high pH or ether and high salt, respectively. The 13cis M-412 absorption spectrum with a maximum at 405 nm [6] corroborated with the action spectrum of a photoactivated M-412 component obtained under sirnil& conditions [7]. Raman spectra had been explained in terms of 13-cis [8,9] and also in terms of all-trans M-41 2 [lo]; in [ 1 l] Raman spectra with 15deuteroretinal indicated that the M-412 Raman spectra originated from the 13-cis isomer of retinal. Furthermore, 13cis retinal was extracted from both M-41 2 and L-550 [ 121, and 13&s retinal extracted from M-412 could be correlated with the amount of recycling bR [13]. are expected. We found reversible absorption changes between 300 and 375 nm as well as between 360 and 450 nm which clearly indicates the appearance of 13cis and the disappearance of all-trans bands of the retinal chromophore reversibly during the all-trans photocycle*.

Properties of strange attractors in yeast glycolysis

The properties of periodic and aperiodic glycolytic oscillations observed in yeast extracts under sinusoidal glucose input were analyzed by the following methods. (1) Spectral analysis, rendering sharp peaks for periodic responses and enhanced broad-band noise for aperiodic oscillations. (2) Phase plane analysis, leading to closed and to open trajectories for periodic and aperiodic oscillations, respectively. (3) Rotation of a phase plane proportionally to time, revealing strange attractors associated with the aperiodic oscillations. (4) Stroboscopic plot on the phase plane, showing that the strange attractors follow a stretch-fold-press process, if the stroboscoping phase is varied. (5) Stroboscopic transfer plot, admitting a period of three transfer processes and thus implying chaos according to the Li-Yorke theorem. (6) Determination of the rate of information production by differentiation of the transfer plot, yielding approx. 0.21 bits per min for the chaotically glycolyzing yeast extract.

Inhibition of yeast mitochondrial F1-ATPase, F0F1-ATPase and submitochondrial particles by rhodamines and ethidium bromide

ATP hydrolysis by F1-ATPase is strongly inhibited by cationic rhodamines; neutral rhodamines are very poor inhibitors. Rhodamine 6G is a noncompetitive inhibitor of purified F0F1-ATPase and submitochondrial particles, however, an uncompetitive inhibitor of F1-ATPase (KI approximately equal to 2.4 microM for all three enzyme forms). Ethidium bromide is a noncompetitive inhibitor of F0F1-ATPase, submitochondrial particles and also F1-ATPase (KI approximately equal to 270 microM). Neither of the inhibitors affects the negative cooperativity (nH approximately equal to 0.7). The non-identical binding sites for rhodamine 6G and ethidium bromide are located on the F1-moiety and are topologically distinct from the catalytic site. Binding of the inhibitors prevents the conformational changes essential for energy transduction. It is concluded that the inhibitor binding sites are involved in proton translocation. In F1-ATPase, binding of MgATP at a catalytic site causes conformational changes, which allosterically induce the correct structure of the rhodamine 6G binding site. In F0F1-ATPase, this conformation of the F1-moiety exists a priori, due to allosteric interactions with F0-subunits. The binding site for ethidium bromide on F1-ATPase does not require substrate binding at the catalytic site and is not affected by F0F1-subunit interactions.

Dynamic Coupling and Time-Patterns of Glycolysis

The property of self-organization is a fundamental feature of living systems. Macroscopically, it is reflected in the phenomena of evolution, of differentiation and of numerous other biological functions. The quality of organization results from basic thermodynamic and kinetic constraints, to which the occurrence of biological systems is fundamentally bound, and its theoretical frame lies in the concept of dissipative structure as a new science of motion.

[32] Spectroscopic methods for protonation state determination

Publisher Summary This chapter discusses various spectroscopic methods for the determination of the protonation state of the purple membrane during the photocycle of the retinal chromophore for steady-state and transient-state conditions. The analysis of the aromatic spectral region indicates deprotonation of tyrosine and proton displacement from tryptophan. The simultaneous measurement of the protons released into the medium—using the pH indicator methylumbelliferone (MU)—and of the state of the photocycle allows a quantification of the number of protons related to each photocycle. The purple membrane of Halobacterium halobium acts as a light-driven proton pump located at the bacteriorhodopsin molecule. The spectroscopic study of the photocycle as well as the cycle of deprotonation and protonation establishes the ratio between the number of protons turned over and the turnover of the photocycle. The spectroscopic analysis of the deprotonation–protonation cycle requires intrinsic or extrinsic indicators that optically—by absorption or fluorescence—respond to proton dissociation reactions.

Interaction between the retinal chromophore and amino acid residues in bacteriorhodopsin

The transition from a noncrystalline to a crystalline protein state when retinal binds to bacterio-opsin, and the spectral shift of the retinal-Schiff-base linkage to 570 nm, indicate strong interaction between the retinal chromophore and amino acid side chains of the protein in bacteriorhodopsin (bR) (I). In order to define the physico-chemical nature of these proteinchromophore interactions we have investigated: a. the bphaviour of proton binding groups and aromatic groups of the protein upon light activation of the retinal chromophore and b. the basic structural requirements for such an interaction by testing chemically sythesized peptides with sequences containing the retinal binding site for their interaction with retinal.