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The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.
Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).
The core of photosystem I (PS1) is composed of the two related integral membrane polypeptides, PsaA and PsaB, which bind two symmetrical branches of cofactors, each consisting of two chlorophylls and a phylloquinone, that potentially link the primary electron donor and the tertiary acceptor. In an effort to identify amino acid residues near the phylloquinone binding sites, all tryptophans and histidines that are conserved between PsaA and PsaB in the region of the 10th and 11th transmembrane alpha-helices were mutated in Chlamydomonas reinhardtii. The mutant PS1 reaction centers appear to assemble normally and possess photochemical activity. An electron paramagnetic resonance (EPR) signal attributed to the phylloquinone anion radical (A(1)(-)) can be observed either transiently or after illumination of reaction centers with pre-reduced iron-sulfur clusters. Mutation of PsaA-Trp(693) to Phe resulted in an inability to photo-accumulate A(1)(-), whereas mutation of the analogous tryptophan in PsaB (PsaB-Trp(673)) did not produce this effect. The PsaA-W693F mutation also produced spectral changes in the time-resolved EPR spectrum of the P(700)(+) A(1)(-) radical pair, whereas the analogous mutation in PsaB had no observable effect. These observations indicate that the A(1)(-) phylloquinone radical observed by EPR occupies the phylloquinone-binding site containing PsaA-Trp(693). However, mutation of either tryptophan accelerated charge recombination from the terminal Fe-S clusters.
The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.
The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports SQ bound in the vicinity of reduced bH heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR-silent SQ bound close to oxidized bH, we calculated a midpoint redox potential (Em) of approximately 90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ.bH(3+) complexes. This favors recycling of the electron coming from center P and also implies a >2.5-fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre-steady-state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ.bH(3+) formation without inhibition of Q binding by QH2.
We have investigated the mechanism responsible for half-of-the-sites activity in the dimeric cytochrome bc(1) complex from Paracoccus denitrificans by characterizing the kinetics of inhibitor binding to the ubiquinol oxidation site at center P. Both myxothiazol and stigmatellin induced a 2-3 nm shift of the visible absorbance spectrum of the b(L) heme. The shift generated by myxothiazol was symmetric, with monophasic kinetics that indicate equal binding of this inhibitor to both center P sites. In contrast, stigmatellin generated an asymmetric shift in the b(L) spectrum, with biphasic kinetics in which each phase contributed approximately half of the total magnitude of the spectral change. The faster binding phase corresponded to a more symmetrical shift of the b(L) spectrum relative to the slower binding phase, indicating that approximately half of the center P sites bound stigmatellin more slowly and in a different position relative to the b(L) heme, generating a different effect on its electronic environment. Significantly, the slow stigmatellin binding phase was lost as the inhibitor concentration was increased. This implies that a conformational change is transmitted from one center P site in the dimer to the other upon stigmatellin binding to one monomer, rendering the second site less accessible to the inhibitor. Because the position that stigmatellin occupies at center P is considered to be analogous to that of the quinol substrate at the moment of electron transfer, these results indicate that the productive enzyme-substrate configuration is prevented from occurring in both monomers simultaneously.
We previously proposed that the dimeric cytochrome bc(1) complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc(1) monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289-22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc(1) complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10-16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c(1) by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.
Highlights
• USP32 deubiquitinates the Ragulator complex subunit LAMTOR1 at lysine (K) 20
• LAMTOR1 K20 ubiquitination impairs its binding to the vacuolar H+-ATPase
• USP32 knockout reduces mTORC1 activity and elevates autophagic flux
• Depletion of USP32 in Caenorhabditis elegans inhibits mTOR and induces autophagy
Summary
The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain–containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63-, and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez. Our results indicate that UBACez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.
Die Bedeutung der Toleranztests für die Diagnostik der diabetischen Vorstadien wird aus den pathophysiologischen Voraussetzungen abgeleitet. Die Methode der Wahl ist der einzeitige orale Glucosetoleranztest mit 100g Glucose (oder Glucoseoligosacchariden). Dieses Verfahren erfaßt die wesentlichen physiologisch wichtigen Funktionen, was für den intravenösen Glucosetoleranztest und für den Tolbutamidtest nicht gilt. Gleichzeitig ist der orale Toleranztest am einfachsten durchzuführen. Lediglich bei Störungen im Gastrointestinalbereich kann der intravenöse Glucosetoleranztest angezeigt sein. Es gibt keine wichtige Indikation für den Tolbutamidtest.
Die verschiedenen Störmöglichkeiten sowie Fehlermöglichkeiten bei der Durchführung des Glucosetoleranztests werden besprochen.