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1.
 The role of the polypeptide matrix in electron transfer processes in proteins has been studied in two distinct systems: first in a protein where the induced ET is artificial, and second as part of the catalytic cycle of an enzyme. Azurins are structurally well-characterized blue single-copper proteins consisting of a rigid β-sheet polypeptide matrix. We have determined rate constants and activation parameters for intramolecular long-range electron transfer between the disulfide radical anions (generated by pulse radiolysis) and the copper(II) centre as a function of driving force and nature of the intervening medium in a large number of wild-type and single-site-mutated proteins. In ascorbate oxidase, for which the three-dimensional structure is equally well characterized, the internal ET from the type-I Cu(I) to the trinuclear Cu(II) centre has been studied. We find that the results correlate well with distance through well-defined pathways using a through-bond electron tunnelling mechanism. Received: 2 January 1997 / Accepted: 6 February 1997  相似文献   
2.
Experimental data were combined with computational methods in constructing a hypothetical three-dimensional model for the blue single copper protein Rhus stellacyanin (St). The known sequence of stellacyanin and its homology with plastocyanin (Pc) were used together with the results of spectroscopic studies of the protein that yielded the current assignment of two histidines, one cysteine and a disulfide sulfur as copper ligands in stellacyanin. By computer graphics and energy minimization the folding of the protein was predicted. The model structure is somewhat less regular than Pc as judged by surface area and energy comparisons, but it is a stable structure. Besides rotation of one imidazole ring the copper site undergoes no change even in the absence of the copper ion and the model shows that the site can be constructed with the four assumed copper ligands without forming a strained system. The structure also indicates that a carbonyl oxygen atom is near the copper, thus the site may have analogy to the Alcaligenes denitrificans azurin (Az) site, although the amino acid sequence is more homologous to that of Pc. The model indicates that aspartate 49, reductively labeled by Cr(III), is near the copper center and homologous to the site labeled by Cr(III) on Pc. Also homologous to Pc is a tyrosine residue adjacent to the aspartate. This tyrosine has been implicated in Pc electron transfer and thus is probably involved in electron transfer reactivity of St as well. The higher reactivity of St with small-molecule redox reagents compared to Az and Pc, may be due to the proximity of the above-mentioned aspartate 49 to the Cu, or the greater exposure of one of the Cu cysteine ligands, in the predicted structure as compared to that in the known Pc and Az structures.  相似文献   
3.
An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.  相似文献   
4.
In contrast to European countries, the overwhelming majority of dogs in the U.S. are neutered (including spaying), usually done before one year of age. Given the importance of gonadal hormones in growth and development, this cultural contrast invites an analysis of the multiple organ systems that may be adversely affected by neutering. Using a single breed-specific dataset, the objective was to examine the variables of gender and age at the time of neutering versus leaving dogs gonadally intact, on all diseases occurring with sufficient frequency for statistical analyses. Given its popularity and vulnerability to various cancers and joint disorders, the Golden Retriever was chosen for this study. Veterinary hospital records of 759 client-owned, intact and neutered female and male dogs, 1–8 years old, were examined for diagnoses of hip dysplasia (HD), cranial cruciate ligament tear (CCL), lymphosarcoma (LSA), hemangiosarcoma (HSA), and mast cell tumor (MCT). Patients were classified as intact, or neutered early (<12 mo) or late (≥12 mo). Statistical analyses involved survival analyses and incidence rate comparisons. Outcomes at the 5 percent level of significance are reported. Of early-neutered males, 10 percent were diagnosed with HD, double the occurrence in intact males. There were no cases of CCL diagnosed in intact males or females, but in early-neutered males and females the occurrences were 5 percent and 8 percent, respectively. Almost 10 percent of early-neutered males were diagnosed with LSA, 3 times more than intact males. The percentage of HSA cases in late-neutered females (about 8 percent) was 4 times more than intact and early-neutered females. There were no cases of MCT in intact females, but the occurrence was nearly 6 percent in late-neutered females. The results have health implications for Golden Retriever companion and service dogs, and for oncologists using dogs as models of cancers that occur in humans.  相似文献   
5.
6.
The chemistry of Type 2 copper depleted T2D Rhus laccase has been investigated with regard to the binding of peroxide, and the ability of the enzyme to undergo reduction and reoxidation. Although the peroxide affinity is diminished in the T2D enzyme (104 M?1) relative to the holo-enzyme ((? 108 M?1) the actual mode of binding as a Type 3 μ-peroxo complex remains, as indicated by absorption and CD spectral measurements. Anaerobic reductive and reoxidative titrations with hydroquinone and hydrogen peroxide respectively revealed that the Type 3 copper pairwise interaction is disrupted during reduction but can be restored on reoxidation. The concept of separate Type 2 and Type 3 copper redox centers is suggested to be inadequate in view of the loss of functional integrity by the Type 3 site on removal of Type 2 copper.  相似文献   
7.
Farver O  Eady RR  Sawers G  Prudêncio M  Pecht I 《FEBS letters》2004,561(1-3):173-176
Pulse radiolysis has been employed to investigate the intramolecular electron transfer (ET) between the type 1 (T1) and type 2 (T2) copper sites in the Met144Ala Alcaligenes xylosoxidans nitrite reductase (AxCuNiR) mutant. This mutation increases the reduction potential of the T1 copper center. Kinetic results suggest that the change in driving force has a dramatic influence on the reactivity: The T2Cu(II) is initially reduced followed by ET to T1Cu(II). The activation parameters have been determined and are compared with those of the wild-type (WT) AxCuNiR. The reorganization energy of the T2 site in the latter enzyme was calculated to be 1.6+/-0.2 eV which is two-fold larger than that of the T1 copper center in the WT protein.  相似文献   
8.
Bis(monoacylglycero)phosphate (BMP) is an endosomal lipid with a unique structure that is implicated in the formation of intraendosomal vesicular bodies. Here we have characterized the effects of dioleoyl-BMP (BMP18:1) at concentrations of 5, 10, 15 and 20 mol% on the thermotropic behavior of dipalmitoyl phosphatidylcholine (DPPC) vesicles, and compared them to those of equimolar concentrations of dioleoyl phosphatidylglycerol (DOPG), a structural isoform of BMP18:1. Because BMP is found in the acidic environments of the late endosome and intralysosomal vesicles, samples were prepared at pH 4.2 to mimic the pH of the lysosome. Both 2H NMR of perdeuterated DPPC and spin-labeled EPR with 16-doxyl phosphatidylcholine were utilized in these investigations. NMR and EPR results show that BMP18:1 induces a lowering in the main phase transition temperature of DPPC similar to that of DOPG. The EPR studies reveal that BMP18:1 induced more disorder in the Lβ phase when compared to equimolar concentrations of DOPG. Analysis from dePaked 2H NMR spectra in the Lα phase reveals that BMP18:1 induces less disorder than equal concentrations of DOPG. Additionally, the results demonstrate that BMP mixes with other phospholipids as a phospholipid and not as a detergent molecule as once speculated.  相似文献   
9.
Xu Z  Farver W  Kodukula S  Storch J 《Biochemistry》2008,47(42):11134-11143
Niemann-Pick disease type C (NPC) is caused by defects in either the NPC1 or NPC2 gene and is characterized by accumulation of cholesterol and glycolipids in the late endosome/lysosome compartment. NPC2 is an intralysosomal protein that binds cholesterol in vitro. Previous studies demonstrated rapid rates of cholesterol transfer from NPC2 to model membranes [Cheruku, S. R., et al. (2006) J. Biol. Chem. 281, 31594-31604]. To model the potential role of NPC2 as a lysosomal cholesterol export protein, in this study we used fluorescence spectroscopic approaches to examine cholesterol transfer from membranes to NPC2, assessing the rate, mechanism, and regulation of this transport step. In addition, we examined the effect of NPC2 on the rate and kinetic mechanism of intermembrane sterol transport, to model the movement of cholesterol from internal lysosomal membranes to the limiting lysosomal membrane. The results support the hypothesis that NPC2 plays an important role in endo/lysosomal cholesterol trafficking by markedly accelerating the rates of cholesterol transport. Rates of sterol transfer from and between membranes were increased by as much as 2 orders of magnitude by NPC2. The transfer studies indicate that the mechanism of NPC2 action involves direct interaction of the protein with membranes. Such interactions were observed directly using FTIR spectroscopy and protein tryptophan spectral shifts. Additionally, cholesterol transfer by NPC2 was found to be greatly enhanced by the unique lysosomal phospholipid lyso-bisphosphatidic acid (LBPA), suggesting an important role for LBPA in NPC2-mediated cholesterol trafficking.  相似文献   
10.
We previously reported an altered hyaluronan (HA) metabolism in idiopathic pulmonary arterial hypertension (IPAH) lung tissue and cultured smooth muscle cells. Hyaluronan was present in the smooth muscle cell layer surrounding the pulmonary vasculature and in plexigenic lesions. Additionally, cultured pulmonary artery smooth muscle cells produced spontaneous HA “cable” structures, without additional stimuli, that were leukocyte-adhesive. We now present evidence that the HA that accumulates in IPAH plexigenic lesions is a pathological form of HA in which heavy chains (HCs) from the serum-derived proteoglycan inter-α-inhibitor are covalently attached to the HA backbone to form a pathological HC-HA complex. CD45-positive leukocytes were identified within these HC-HA matrices. Elevated mRNA levels of the enzyme that transfers HCs to HA, known as tumor necrosis factor-stimulated gene 6, were detected in IPAH lung tissue.  相似文献   
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