Stanley Seltzer

Catalyzed cis-trans isomerization of retinals by crude tissue extracts

Abstract Tissue extracts in a neutral anaerobic environment have previously been shown to catalyze 11- cis-retinal → all- trans + 13- cis-retinal trans-retinal to the 9-cis isomer. The latter reaction results in the formation of artifactual isorhodopsin. We find that in an aerobic, mildly alkaline environment, however, a reaction of contrasting specificity is observed. While 11- cis-retinal → all- trans + 13- cis-retinal, 9- cis is found to equilibrate with 9, 13- dicis-retinal. No 9- cis is formed from trans or 13- cis-retinals. A rationale is given for the predominant reactivity about the C-13 double bond of retinals in the presence of this apparent high molecular weight protein(s) catalyst.

Maleylacetoacetate cis-trans isomerase: One-step double cis-trans isomerization of monomethyl muconate and the enzyme's probable role in benzene metabolism

Abstract Maleylacetoacetate cis-trans isomerase together with glutathione has been found to isomerize cis-trans isomers of monomethyl muconate. Isomerization about a single double bond and concerted double isomerization of the diene unit occurs. In addition to the variations in substrate structure previously identified the current results demonstrate that a cis,cis diene skeleton and a conjugated ester function are accepted by the enzyme. The present work and the fiding of trans,trans -muconic acid in the urine of benzene-fed mice ( M. M. Gad-El-Karim, V. M. Sadagopa Ramanujam, and M. S. Legator (1985) Xenobiotica 15 , 211) suggest that maleylacetoacetate cis-trans isomerase may be responsible for the geometrical isomerization. However, cis,cis -muconaldehydic acid rather than cis,cis -muconic acid is suggested to be the early intermediate in benzene metabolism capable of rapid enzyme-catalyzed cis-trans isomerization.

Light-dependent nitration of bacteriorhodopsin.

Purple membranes were treated with tetranitromethane to modify tyrosine residues of bacteriorhodopsin. At pH 8.0, nitration is shown to be affected by illumination during the modification. Amino acid analysis revealed about 0.7 residues nitrated if reaction was in the dark while about 2.0 tyrosines were modified if illumination greater than 540 nm was provided. Tryptophan was unaffected under both conditions. Light-dependent nitration caused a blue shift of the absorbance maximum of bacteriorhodopsin from 568 to 530 nm while no chromophore shift was observed for the dark-modified preparation. Both preparations show an absorption band at 360 nm indicative of the presence of nitrotyrosines. Reduction by dithionite eliminated the pH-dependent changes associated with the 360-nm nitrotyrosine band. Circular dichroism spectra indicate that interactions between neighboring chromophores are altered concomitant with the blue shift of the absorbance maximum by nitration. These studies show that light is required for the nitration of the tyrosine residue, and that Tyr 26 (H. D. Lemke and D. Oesterhelt (1981) Eur. J. Biochem. 115, 595-604) is probably responsible for the blue shift of the absorbance maximum. The intrinsic fluorescence and photocycle kinetics of the tyrosine-modified preparation and reduction of nitrotyrosine by dithionite were studied. In dark modification, only pH-dependent dithionite-reducible nitrotyrosines were produced. It is concluded that surface tyrosines probably do not directly participate in the proton-translocation events coupled to the photocycle of bacteriorhodopsin.

Maleylacetone cis-trans isomerase: formation of an N-ethylmaleimide-labeled enzyme only during the slow phase of the biphasic inhibition reaction

Previous reports from this laboratory have discussed the mechanism of GSH-interaction with the substrate in the enzymatic reaction [2,3]. The enzyme which is highly labile to air oxidation, can be protected with mercaptoethanol and/or EDTA. The isomerase has been reported to be irreversibly inactivated by NEM and hence believed to have an important thiol group [ 11. Here, we show that the kinetics of NEM-inactivation is biphasic. Covalent attachment of the inhibitor to the only cysteinyl thiol occurs in the slower phase. No covalent attachment of NEM in the rapid phase can be detected.

[Mesityl]bacteriorhodopsin. The properties of an analogue of the purple membrane containing [mesityl]retinal as the chromophore

Abstract— A new synthesis of all‐trans‐[mesityl]retinal, II, (all‐trans‐3,7‐dimethyl‐9‐(2′,4′,6′‐trimethylphenyl)‐2,4,6,8,‐nonatetraenal) and 13‐cis‐[mesityl]retinal, VI, (3,7‐dimethyl‐9‐(2′4′6′‐trimethylphenyl)‐2Z,4E,6E,8E‐nonatetraenal) is reported. Combination of all‐trans‐[mesityl]retinal with bacterioopsin results in the formation of a synthetic membrane (Λmax 460) which has photocycling properties similar to the purple membrane although its cycling rate is very much slower. An M‐type intermediate can be trapped at ‐60°C. Photoreversal of the M‐intermediate to the wavelength of initial absorption is observed. Phototransformation of the initial [mesityl]bacteriorhodopsin is accompanied by conversion of the all‐frans to the 13‐cis‐isomer.

THE PURPLE MEMBRANE PROTON PUMP: A MECHANISTIC PROPOSAL FOR THE SOURCE OF THE SECOND PROTON*

Abstract— A mechanistic proposal is presented to account for the observation that two protons are pumped for each turnover in the photocycle of bacteriorhodopsin. One of the protons pumped presumably arises from the deprotonation of the Schiff base as suggested by others. Photoexcitation of retinal is known to result in substantial positive charge development in the six membered ring of the chromophore. It is suggested that the other proton pumped comes from a proteinic Brönsted acid group located close to the cyclohexenyl ring of the bound chromophore. Calculations indicate that the acidity of such a suitably placed Brönsted acid can be increased 3 to 5.5 pK units by virtue of a purely electrostatic effect if 0.2 units of additional positive charge develops atC–5 of the chromophore in its excited state. Two other observations: (a) the bathochromic shift and (b) the deuterium isotope effect in the formation of the first intermediate (bR568→hv K610) are also shown to be consistent with this mechanistic proposal.

A HIGHLY REACTIVE HETEROATOM ANALOG OF RETINAL AND ITS INTERACTION WITH BACTERIORHODOPSIN

Abstract

Secondary. beta. -deuterium isotope effect in the formation of ethyl radical from decomposition of methylethylethyl-2,2,2-d/sub 3/-carbinyloxy radical

The intramolecular secondary ..beta..-deuterium isotope effect in the decomposition of methylethylethyl-2,2,2-d/sub 3/-carbinyloxy radical to methyl ethyl ketone and ethyl radical is k/sub H/sub 3///k/sub D/sub 3// = 1.25 and is relatively temperature independent between 0 and 80/sup 0/C. The comparison of the observed effect with previous theoretical predictions is discussed as well as the predicted temperature dependence. (auth)

The secondary β-deuterium isotope effect in dark adaptation of bacteriorhodopsin containing retinal-20,20,20-d3

Abstract The secondary deuterium isotope effect on the rate of dark adaptation of photobleached purple membrane reconstituted with retinal- 20,20,20-d 3 is k H k D = 0.87 ± 0.02 . The inverse isotope effect which reflects the change in hyperconjugative stabilization of positive charge at retinals C13 in going from reactant to the transition state of the rate-controlling step is rationalized, with the aid of MNDO calculations, as being due to the addition of a nucleophile, presumably the carboxylate of aspartate-212, to retinals C13 to catalyze the double cis-trans isomerization which is attendant with the dark-adaptation process.

The consequences of a deuterium exchange test on proposed mechanisms for the purple membrane proton pump

The capture of radiant energy from the sun by Halobacterium halobium and its ability to use this energy to power its proton pump [l-3] has received much attention recently. Their energy converters, which are purple patches that the bacteria develop in their cell membrane when the dioxygen concentration of the growth medium is reduced, are essentially 75% protein and the rest lipid [4]. The purple pigment, bacteriorhodopsin (bR), results from a combination of retinal and bacterioopsin (mol. wt 26 000), through a Schiff base linkage with a lysine residue [4,5]. It has been shown that the chromophore of light-adapted bR is all-tram retinal while that of the dark-adapted contains equal amounts of 134s and all-tram retinal [3]. Photoexcitation of bacteriorhodopsin @,, 570 nm) produces a red-shifted intermediate, bathobacteriorhodopsin(h,, 610 nm), as the first detectable species [6]. A similar intermediate is formed in the rhodopsin system. Because this initial transformation in rhodopsin [7,8] and bacteriorhodopsin [8] has a low activation energy and exhibits a kinetic isotope effect in D20, it has been suggested that a proton transfer is involved. The Schiff base nitrogen has been proposed as the proton acceptor in rhodopsin [7-91 and implied for bacteriorhodopsin [8]. An alternative view, however, suggests that the formation of the red-shifted intermediate is due to a protein conformational change in the retinal binding region and that the isotope effect results from the attendant proton transfers within the protein [lo].