共查询到19条相似文献,搜索用时 15 毫秒
1.
Kuninori Suzuki Chika Kondo Mayumi Morimoto Yoshinori Ohsumi 《The Journal of biological chemistry》2010,285(39):30019-30025
In Saccharomyces cerevisiae, aminopeptidase I (Ape1p) and α-mannosidase (Ams1p) are known cargoes of selective autophagy. Atg19p has been identified as an Ape1p receptor and targets Ape1p to the preautophagosomal structure (PAS). Under nutrient-rich conditions, transport of Ams1p to the vacuole largely depends on Atg19p. Here, we show that Atg34p (Yol083wp), a homolog of Atg19p, is a receptor for Ams1p transport during autophagy. Atg34p interacted with Ams1p, Atg11p, and Atg8p using distinct domains. Homo-oligomerized Ams1p bound to the Ams1-binding domain of Atg34p; this binding was important for the formation of a higher order complex named the Ams1 complex. In the absence of the interaction of Atg34p with Atg8p, the Ams1 complex was targeted to the preautophagosomal structure but failed to transit to the vacuole, indicating that the interaction of Atg34p with Atg8p is crucial for the Ams1 complex to be enclosed by autophagosomes. Atg34p and Atg19p have similar domain structures and are important for Ams1p transport during autophagy. 相似文献
2.
Lu Huo Ian Davis Lirong Chen Aimin Liu 《The Journal of biological chemistry》2013,288(43):30862-30871
Although the crystal structure of α-amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase from Pseudomonas fluorescens was solved as a dimer, this enzyme is a mixture of monomer, dimer, and higher order structures in solution. In this work, we found that the dimeric state, not the monomeric state, is the functionally active form. Two conserved arginine residues are present in the active site: Arg-51 and an intruding Arg-239* from the neighboring subunit. In this study, they were each mutated to alanine and lysine, and all four mutants were catalytically inactive. The mutants were also incapable of accommodating pyridine-2,6-dicarboxylic acid, a competitive inhibitor of the native enzyme, suggesting that the two Arg residues are involved in substrate binding. It was also observed that the decarboxylase activity was partially recovered in a heterodimer hybridization experiment when inactive R51(A/K) and R239(A/K) mutants were mixed together. Of the 20 crystal structures obtained from mixing inactive R51A and R239A homodimers that diffracted to a resolution lower than 3.00 Å, two structures are clearly R51A/R239A heterodimers and belong to the C2 space group. They were refined to 1.80 and 2.00 Å resolutions, respectively. Four of the remaining crystals are apparently single mutants and belong to the P42212 space group. In the heterodimer structures, one active site is shown to contain dual mutation of Ala-51 and Ala-239*, whereas the other contains the native Arg-51 and Arg-239* residues, identical to the wild-type structure. Thus, these observations provide the foundation for a molecular mechanism by which the oligomerization state of α-amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase could regulate the enzyme activity. 相似文献
3.
Christopher I. Richards Rahul Srinivasan Cheng Xiao Elisha D. W. Mackey Julie M. Miwa Henry A. Lester 《The Journal of biological chemistry》2011,286(36):31241-31249
We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few α4β4 nAChRs, but many α4β2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the β4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of α4β4 receptor-containing vesicles with the PM by ∼2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2–3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of α4β4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 μm) did not alter the PM insertion frequency or trafficking behavior of α4β4-laden vesicles. In contrast, chronic nicotine substantially increased the number of α4β2-containing vesicle fusions at the PM; this stage in α4β2 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction. 相似文献
4.
5.
6.
Determination of α2HS-glycoprotein phenotypes by isoelectric focusing and immunoblotting: polymorphic occurrence of HSGA
*5 in Okinawa 总被引:1,自引:0,他引:1
Summary A print immunofixation is a very useful procedure for the demonstration of 2HS-glycoprotein (HSGA) following polyacrylamide gel isoelectric focusing. However, this technique has the one disadvantage of requiring a large volume of expensive antiserum. In this paper an alternative detection system is presented which involves the non-electric transfer of HSGA from a focused gel to a nitrocellulose filter and the immunologic detection of HSGA immobilized on nitrocellulose. Using this method the distribution of HSGA polymorphism in the Nepalese and Japanese populations was investigated. All the populations tested were found to lack the common HSGA
*3 of the Caucasians. A rare HSGA
*5 in Honshu, a main island of Japan, was observed at polymorphic frequency in Okinawa, Southern Japan. 相似文献
7.
Roland Baur Kuldeep H. Kaur Erwin Sigel 《The Journal of biological chemistry》2010,285(23):17398-17405
δ subunit-containing γ-aminobutyric acid, type A (GABAA)receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with α1 and/or α6 subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABAA receptor pentamers by concatenation. These receptors composed of α1, α6, β3, and δ subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3α,21-dihydroxy-5α-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that δ can assume multiple positions in a receptor pentamer made up of α1, α6, β3, and δ subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of δ subunits between two α subunits in α1α6β3δ receptors. This property is shared by α1β3δ and α6β3δ receptors, but there are differences in the additionally expressed isoforms. 相似文献
8.
Fauvet B Fares MB Samuel F Dikiy I Tandon A Eliezer D Lashuel HA 《The Journal of biological chemistry》2012,287(34):28243-28262
N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively N(α)-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of N(α)-acetylation on the biophysical and biological properties of α-syn, we produced N(α)-acetylated α-syn first using a semisynthetic methodology based on expressed protein ligation (Berrade, L., and Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909-3922) and then a recombinant expression strategy, to compare its properties to unacetylated α-syn. We demonstrate that both WT and N(α)-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing N(α)-acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both N(α)-acetylated and WT α-syn exhibited similar ability to bind synaptosomal membranes in vitro and in HeLa cells, where both internalized proteins exhibited prominent cytosolic subcellular distribution. We then determined the effect of attenuating N(α)-acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn N(α)-acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells. 相似文献
9.
10.
Vishukumar Aimanianda C��cile Clavaud Catherine Simenel Thierry Fontaine Muriel Delepierre Jean-Paul Latg�� 《The Journal of biological chemistry》2009,284(20):13401-13412
Despite its essential role in the yeast cell wall, the exact composition of
the β-(1,6)-glucan component is not well characterized. While
solubilizing the cell wall alkali-insoluble fraction from a wild type strain
of Saccharomyces cerevisiae using a recombinant
β-(1,3)-glucanase followed by chromatographic characterization of the
digest on an anion exchange column, we observed a soluble polymer that eluted
at the end of the solvent gradient run. Further characterization indicated
this soluble polymer to have a molecular mass of ∼38 kDa and could be
hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry
and NMR (1H and 13C) analyses confirmed it to be a
β-(1,6)-glucan polymer with, on average, branching at every fifth residue
with one or two β-(1,3)-linked glucose units in the side chain. This
polymer peak was significantly reduced in the corresponding digests from
mutants of the kre genes (kre9 and kre5) that are
known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the
current study, we have developed a biochemical assay wherein incubation of
UDP-[14C]glucose with permeabilized S. cerevisiae yeasts
resulted in the synthesis of a polymer chemically identical to the branched
β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters
essential for β-(1,6)-glucan synthetic activity were defined.The cell wall of Saccharomyces cerevisiae and other yeasts
contains two types of β-glucans. In the former yeast, branched
β-(1,3)-glucan accounts for ∼50–55%, whereas
β-(1,6)-glucan represents 10–15% of the total yeast cell wall
polysaccharides, each chain of the latter extending up to 140–350
glucose residues in length. The amount of 3,6-branched glucose residues varies
with the yeast species: 7, 15, and 75% in S. cerevisiae, Candida
albicans, and Schizosaccharomyces pombe, respectively
(1). β-(1,6)-Glucan
stabilizes the cell wall, since it plays a central role as a linker for
specific cell wall components, including β-(1,3)-glucan, chitin, and
mannoproteins (2,
3). However, the exact
structure of the β-(1,6)-glucan and the mode of biosynthesis of this
polymer are largely unknown. In S. pombe, immunodetection studies
suggested that synthesis of this polymer backbone begins in the endoplasmic
reticulum, with extension occurring in the Golgi
(4) and final processing at the
plasma membrane. In S. cerevisiae, Montijn and co-workers
(5), by immunogold labeling,
detected β-(1,6)-glucan at the plasma membrane, suggesting that the
synthesis takes place largely at the cell surface.More than 20 genes, including the KRE gene family (14 members) and
their homologues, SKN1 and KNH1, have been reported to be
involved in β-(1,6)-glucan synthesis in S. cerevisiae, C.
albicans, and Candida glabrata
(6–10).
Among all of these genes, the ones that seem to play the major synthetic role
are KRE5 and KRE9, since their disruption caused significant
reduction (100 and 80%, respectively, relative to wild type) in the cell wall
β-(1,6)-glucan content
(11–13).To date, the biochemical reaction responsible for the synthesis of
β-(1,6)-glucan and the product synthesized remained unknown. Indeed, in
most cases, when membrane preparations are incubated with UDP-glucose, only
linear β-(1,3)-glucan polymers are produced, although some studies have
reported the production of low amounts of β-(1,6)-glucans by membrane
preparations
(14–17).
These data suggest that disruption of the fungal cell prevents or at least has
a strong negative effect on β-(1,6)-glucan synthesis. The use of
permeabilized cells, which allows substrates, such as nucleotide sugar
precursors, to be readily transported across the plasma membrane, is an
alternative method to study in situ cell wall enzyme activities
(18–22).
A number of methods have been developed to permeabilize the yeast cell wall
(23), of which osmotic shock
was successfully used to demonstrate β-(1,3)-glucan and chitin synthase
activities (20,
24). Herein, we describe the
biochemical activity responsible for β-(1,6)-glucan synthesis using
permeabilized S. cerevisiae cells and UDP-[14C]glucose as
a substrate. We also have analyzed the physicochemical parameters of this
activity and chemically characterized the end product and its structural
organization within the mature yeast cell wall. 相似文献
11.
Lu Meng Farhad Forouhar David Thieker Zhongwei Gao Annapoorani Ramiah Heather Moniz Yong Xiang Jayaraman Seetharaman Sahand Milaninia Min Su Robert Bridger Lucas Veillon Parastoo Azadi Gregory Kornhaber Lance Wells Gaetano T. Montelione Robert J. Woods Liang Tong Kelley W. Moremen 《The Journal of biological chemistry》2013,288(48):34680-34698
Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases. 相似文献
12.
Stephen Ma Colin Hockings Khatira Anwari Tobias Kratina Stephanie Fennell Michael Lazarou Michael T. Ryan Ruth M. Kluck Grant Dewson 《The Journal of biological chemistry》2013,288(36):26027-26038
Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins. 相似文献
13.
Xiaoyi Deng Jeongmi Lee Anthony J. Michael Diana R. Tomchick Elizabeth J. Goldsmith Margaret A. Phillips 《The Journal of biological chemistry》2010,285(33):25708-25719
Pyridoxal 5′-phosphate (PLP)-dependent basic amino acid decarboxylases from the β/α-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-Å resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the β/α-barrel domain of one subunit and the β-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the β-barrel domain we refer to as the “specificity helix.” Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu314, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5′-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold. 相似文献
14.
Helen M. Reid Eamon P. Mulvaney Elizebeth C. Turner B. Therese Kinsella 《The Journal of biological chemistry》2010,285(24):18709-18726
The human prostacyclin receptor (hIP) undergoes agonist-induced internalization and subsequent recyclization in slowly recycling endosomes involving its direct physical interaction with Rab11a. Moreover, interaction with Rab11a localizes to a 22-residue putative Rab11 binding domain (RBD) within the carboxyl-terminal tail of the hIP, proximal to the transmembrane 7 (TM7) domain. Because the proposed RBD contains Cys308 and Cys311, in addition to Cys309, that are known to undergo palmitoylation, we sought to identify the structure/function determinants of the RBD, including the influence of palmitoylation, on agonist-induced trafficking of the hIP. Through complementary approaches in yeast and mammalian cells along with computational structural studies, the RBD was localized to a 14-residue domain, between Val299 and Leu312, and proposed to be organized into an eighth α-helical domain (α-helix 8), comprising Val299–Val307, adjacent to the palmitoylated residues at Cys308–Cys311. From mutational and [3H]palmitate metabolic labeling studies, it is proposed that palmitoylation at Cys311 in addition to agonist-regulated deacylation at Cys309 > Cys308 may dynamically position α-helix 8 in proximity to Rab11a, to regulate agonist-induced intracellular trafficking of the hIP. Moreover, Ala-scanning mutagenesis identified several hydrophobic residues within α-helix 8 as necessary for the interaction with Rab11a. Given the diverse membership of the G protein-coupled receptor superfamily, of which many members are also predicted to contain an α-helical 8 domain proximal to TM7 and, often, adjacent to palmitoylable cysteine(s), the identification of a functional role for α-helix 8, as exemplified as an RBD for the hIP, is likely to have broader significance for certain members of the superfamily. 相似文献
15.
P. Patrizia Mangione Gennaro Esposito Annalisa Relini Sara Raimondi Riccardo Porcari Sofia Giorgetti Alessandra Corazza Federico Fogolari Amanda Penco Yuji Goto Young-Ho Lee Hisashi Yagi Ciro Cecconi Mohsin M. Naqvi Julian D. Gillmore Philip N. Hawkins Fabrizio Chiti Ranieri Rolandi Graham W. Taylor Mark B. Pepys Monica Stoppini Vittorio Bellotti 《The Journal of biological chemistry》2013,288(43):30917-30930
Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β2-microglobulin (β2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition. 相似文献
16.
17.
18.
Makoto Taniguchi Kazuyuki Kitatani Tadakazu Kondo Mayumi Hashimoto-Nishimura Satoshi Asano Akira Hayashi Susumu Mitsutake Yasuyuki Igarashi Hisanori Umehara Hiroyuki Takeya Junzo Kigawa Toshiro Okazaki 《The Journal of biological chemistry》2012,287(47):39898-39910
The role of “sphingolipid rheostat” by ceramide and sphingosine 1-phosphate (S1P) in the regulation of autophagy remains unclear. In human leukemia HL-60 cells, amino acid deprivation (AA(−)) caused autophagy with an increase in acid sphingomyleinase (SMase) activity and ceramide, which serves as an autophagy inducing lipid. Knockdown of acid SMase significantly suppressed the autophagy induction. S1P treatment counteracted autophagy induction by AA(−) or C2-ceramide. AA(−) treatment promoted mammalian target of rapamycin (mTOR) dephosphorylation/inactivation, inducing autophagy. S1P treatment suppressed mTOR inactivation and autophagy induction by AA(−). S1P exerts biological actions via cell surface receptors, and S1P3 among five S1P receptors was predominantly expressed in HL-60 cells. We evaluated the involvement of S1P3 in suppressing autophagy induction. S1P treatment of CHO cells had no effects on mTOR inactivation and autophagy induction by AA(−) or C2-ceramide. Whereas S1P treatment of S1P3 overexpressing CHO cells resulted in activation of the mTOR pathway, preventing cells from undergoing autophagy induced by AA(−) or C2-ceramide. These results indicate that S1P-S1P3 plays a role in counteracting ceramide signals that mediate mTOR-controlled autophagy. In addition, we evaluated the involvement of ceramide-activated protein phosphatases (CAPPs) in ceramide-dependent inactivation of the mTOR pathway. Inhibition of CAPP by okadaic acid in AA(−)- or C2-ceramide-treated cells suppressed dephosphorylation/inactivation of mTOR, autophagy induction, and autophagy-associated cell death, indicating a novel role of ceramide-CAPPs in autophagy induction. Moreover, S1P3 engagement by S1P counteracted cell death. Taken together, these results indicated that sphingolipid rheostat in ceramide-CAPPs and S1P-S1P3 signaling modulates autophagy and its associated cell death through regulation of the mTOR pathway. 相似文献
19.
Quinol oxidation at center P of the cytochrome bc1
complex involves bifurcated electron transfer to the Rieske iron-sulfur
protein and cytochrome b. It is unknown whether both electrons are
transferred from the same domain close to the Rieske protein, or if an
unstable semiquinone anion intermediate diffuses rapidly to the vicinity of
the bL heme. We have determined the pre-steady state rate
and activation energy (Ea) for quinol oxidation in
purified yeast bc1 complexes harboring either a Y185F
mutation in the Rieske protein, which decreases the redox potential of the FeS
cluster, or a E272Q cytochrome b mutation, which eliminates the
proton acceptor in cytochrome b. The rate of the bifurcated reaction
in the E272Q mutant (<10% of the wild type) was even lower than that of the
Y185F enzyme (∼20% of the wild type). However, the E272Q enzyme showed the
same Ea (61 kJ mol-1) with respect to the wild
type (62 kJ mol-1), in contrast with the Y185F mutation, which
increased Ea to 73 kJ mol-1. The rate and
Ea of the slow reaction of quinol with oxygen that are
observed after cytochrome b is reduced were unaffected by the E272Q
substitution, whereas the Y185F mutation modified only its rate. The
Y185F/E272Q double mutation resulted in a synergistic decrease in the rate of
quinol oxidation (0.7% of the wild type). These results are inconsistent with
a sequential “movable semiquinone” mechanism but are consistent
with a model in which both electrons are transferred simultaneously from the
same domain in center P.The cytochrome bc1 complex couples the oxidation of a
two-electron carrier molecule of quinol to the movement of protons across the
inner mitochondrial or bacterial membrane. The key reaction in this
energy-conserving mechanism, known as the Q-cycle
(1,
2), is the bifurcation of
electrons at the active site located closer to the positive side of the
membrane, termed center P or Qo site. One of the electrons from
quinol is transferred to a chain of one-electron carriers with relatively high
redox potentials that include the FeS cluster of the Rieske protein and the
hemes of cytochromes c1 and c. The other electron
is donated to the low potential (bL) heme of cytochrome
b, from which it crosses most of the membrane width to the high
potential bH heme, located close to another active site
(center N or Qi site), where quinone is reduced to quinol after two
center P turnovers. Proton release and uptake at each active site are achieved
by taking advantage of the chemistry of quinol and quinone, which can only
stably exist at physiological pH in the protonated and deprotonated forms,
respectively.Critical to the electron bifurcation reaction at center P is the
arrangement of protonatable groups (His181 of the Rieske protein
and Glu272 of cytochrome b) close to the electron
acceptors at opposite sides of the substrate (see
Fig. 1). However, the exact
mechanism of electron bifurcation at center P is still an unresolved issue.
Proposed models have ranged from strictly concerted mechanisms in which both
electrons from quinol are extracted simultaneously
(3,
4) to those that postulate a
highly stabilized semiquinone intermediate
(5). Between these two extremes
are mechanisms that propose the formation of an unstable semiquinone
intermediate after a first electron transfer from quinol to the Rieske protein
(6–8),
which seem to be supported by recent reports that claim to have detected low
concentrations of semiquinone at center P when reoxidation of cytochrome
b is impeded under special conditions
(9,
10). One version of the
unstable semiquinone mechanism proposes that this intermediate diffuses from
the vicinity of the Rieske protein to a location within center P located
closer to the bL heme, which would allow non-rate-limiting
rates of bL reduction to occur even at very low
semiquinone occupancy (11). In
this proposal, the movement of the unstable semiquinone would be allowed by
protonation and rotation of Glu272 in cytochrome b, which
occupies different conformations in crystallographic structures
(Fig. 1)
(11–14).Open in a separate windowFIGURE 1.Electron and proton acceptors involved in quinol oxidation at center
P. Crystallographic structures 1EZV
(12) and 1P84
(13) show stigmatellin
(A) or 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole
(B) bound at center P forming a hydrogen bond to the
His181 residue of the Rieske protein, which is a ligand to the FeS
cluster. The Tyr185 residue in the Rieske protein influences the
Em value of the FeS cluster
(22). On the side pointing to
the bL heme, a bound water molecule is also
hydrogen-bonded to the inhibitor, either to the Glu272 carboxylate
in cytochrome b (A), or to its backbone amino group
(B), when the side chain is rotated toward a water network that
connects to the propionate of the bL heme and to
Arg79 of cytochrome b.An important prediction of the movable semiquinone model
(11) is that mutation of
Glu272 should impede diffusion of the anionic semiquinone, forcing
electron transfer to the bL heme to occur through a longer
distance from the position closer to the Rieske FeS cluster
(15), thereby shifting the
rate-limiting step from the first to the second electron transfer. Although it
has already been reported that different mutations at Glu272
partially slow down quinol oxidation at center P
(15–17),
no effort has been made so far to evaluate whether the rate-limiting step
changes upon inhibition of the deprotonation of quinol (or of a putative
semiquinone intermediate) by mutation of the cytochrome b
Glu272. In the present work, we analyze the energy of activation of
quinol oxidation at center P and show that the rate-limiting step when
Glu272 is mutated to glutamine, although slower, is still
determined by the driving force for electron transfer to the Rieske protein.
We also show that decreasing this driving force enhances the relative
inhibition caused by mutating Glu272, suggesting a tight coupling
of reactions involved in quinol oxidation and deprotonation. In contrast,
reactions with oxygen that bypass the electron bifurcation at center P, which
are likely to involve a semiquinone intermediate, are independent of
Glu272 and go through an energetic barrier different from that of
the bifurcated reaction. We discuss how these results support a mechanism in
which both electron transfer events from quinol to the Rieske protein and the
bL heme occur from the same position and at the same
time. 相似文献