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When applying photochromic switches as functional units in light-responsive materials or devices, an often disregarded yet crucial property is their resistance to fatigue during photoisomerization. In the large family of diarylethene photoswitches, formation of an annulated isomer as a byproduct of the photochromic reaction turns out to prevent the desired high reversibility for many different derivatives. To overcome this general problem, we have synthesized and thoroughly investigated the fatigue behavior of a series of diarylethenes, varying the nature of the hetaryl moieties, the bridging units, and the substituents. By analysis of photokinetic data, a quantification of the tendency for byproduct formation in terms of quantum yields could be achieved, and a strong dependency on the electronic properties of the substituents was observed. In particular, substitution with 3,5-bis(trifluoromethyl)phenyl or 3,5-bis(pentafluorosulfanyl)phenyl groups strongly suppresses the byproduct formation and opens up a general strategy to construct highly fatigue-resistant diarylethene photochromic systems with a large structural flexibility.

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SummaryTransport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of aminoterminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. lO,OOO-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about l&fold more abundant in mitochondria than MPP It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of "signal peptidase,' different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.

Some rare indigo derivatives have been known for a long time to be photochromic upon irradiation with red light, which should be advantageous for many applications. However, the absence of strategies to tune their thermal half-lives by modular molecular design as well as the lack of proper synthetic methods to prepare a variety of such molecules from the parent indigo dye have so far precluded their use. In this work, several synthetic protocols for N-functionalization have been developed, and a variety of N-alkyl and N-aryl indigo derivatives have been prepared. By installation of electron-withdrawing substituents on the N-aryl moieties, the thermal stability of the Z-isomers could be enhanced while maintaining the advantageous photoswitching properties upon irradiation with red light (660 nm LED). Both experimental data and computational results suggest that the ability to tune thermal stability without affecting the dyes' absorption maxima originates from the twisted geometry of the N-aryl groups. The new indigo photoswitches reported are expected to have a large impact on the development of optical methods and applications in both life and material sciences.

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocationThe great majority of mitochondrial proteins are coded for by nuclear genes. They are synthesized on cytoplasmic ribosomes as precursor proteins, most but not all of which have higher apparent molecular weights than the mature proteins. These precursor proteins are imported into the mitochondria in a posttranslational event [I -1 I].The posttranslational transfer in vivo and in vitro of most, though not all, mitochondrial proteins has been shown to be dependent on the energization of the mitochondria [12-151. In this study we have investigated which form of energy is required for the transfer of proteins into the inner mitochondrial membrane. We have employed various uncouplers, ionophores, and inhibitors of oxidative phosphorylation, ADP/ATP translocation and electron transport to decide whether the direct force for import is the membrane potential or the intramitochondrial ATP. Two proteins of the inner mitochondrial membrane were studied : the ADP/ATP carrier, which is not proteolytically processed during transfer into mitochondria [4], and the dicyclohexylcarbodiimide-binding subunit of oligomycin-sensitive ATPase (subunit 9 of 'proteolipid'), which undergoes proteolytic cleavage [14.16].All results obtained are compatible with the view that it is the membrane potential which is required for protein transfer, and that ATP in the mitochondrial matrix is not the direct energy source.

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Subunit 9 (dicyclohexylcarbodiimide binding protein, 'proteolipid') of the mitochondrial F lFo-ATPase is a nuclearly coded protein in Neurospora crassa. It is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved off after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2 + for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two cleavage sites in the precursor molecule were determined. The data indicate that: (a) the correct NH2-terminus of the mature protein was generated, (b) the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleucine in position -31. The cleavage sites show similarity of primary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NH2-terminal part of these polypeptides) into the matrix space of mitochondria.The majority of mitochondrial proteins are encoded by nuclear genes [l]. They are synthesized on free cytoplasmic ribosomes as water-soluble species, i.e. precursor proteins [2]. In many cases these precursor forms carry NH,-terminal peptide extensions not present in the mature proteins [3]. The free precursors are then selectively transported to their functional sites within the mitochondria. Transport is accompanied by one or more processing steps which may include covalent modifications of precursor proteins and/or proteolytic removal of peptide extensions. The assembly of cytochromes c and c1 requires the covalent attachment of the heme group [4-61. In a few cases proteolytic processing takes place in two steps, as has been shown for cytochrome c1 in Neurospora and yeast and for cytochrome b2 in yeast [5, 61. The mitochondrial enzymes involved in processing have not been extensively characterized. Proteolytic activities have been detected in extracts of mitochondria from different sources [7 -91, which can remove the NH2-terminal peptide extensions or at least part of them from precursor proteins. A partial purification of this activity from yeast mitochondria has been achieved [ 101. The processing activities investigated so far have several features in common: they appear to be soluble components of the mitochondrial matrix and they require divalent metal ions such as Mn2' or Zn2+ for full activity. The processing activity responsible for the second cleavage ...