Na,K-ATPase in skeletal muscle: two populations of beta-spectrin control localization in the sarcolemma but not partitioning between the sarcolemma and the transverse tubules.

We used immunological approaches to study the factors controlling the distribution of the Na,K-ATPase in fast twitch skeletal muscle of the rat. Both alpha subunits of the Na,K-ATPase colocalize with beta-spectrin and ankyrin 3 in costameres, structures at the sarcolemma that lie over Z and M-lines and in longitudinal strands. In immunoprecipitates, the alpha1 and alpha2 subunits of the Na,K-ATPase as well as ankyrin 3 associate with beta-spectrin/alpha- fodrin heteromers and with a pool of beta-spectrin at the sarcolemma that does not contain alpha-fodrin. Myofibers of mutant mice lacking beta-spectrin (ja/ja) have a more uniform distribution of both the alpha1 and alpha2 subunits of the Na,K-ATPase in the sarcolemma, supporting the idea that the rectilinear sarcomeric pattern assumed by the Na,K-ATPase in wild-type muscle requires beta-spectrin. The Na,K-ATPase and beta-spectrin are distributed normally in muscle fibers of the nb/nb mouse, which lacks ankyrin 1, suggesting that this isoform of ankyrin is not necessary to link the Na,K-ATPase to the spectrin-based membrane skeleton. In immunofluorescence and subcellular fractionation experiments, the alpha2 but not the alpha1 subunit of the Na,K-ATPase is present in transverse (t-) tubules. The alpha1 subunit of the pump is not detected in increased amounts in the t-tubules of muscle from the ja/ja mouse, however. Our results suggest that the spectrin-based membrane skeleton, including ankyrin 3, concentrates both isoforms of the Na,K-ATPase in costameres, but that it does not play a significant role in restricting the entry of the alpha1 subunit into the t-tubules.

Hematopoietic cells from -spectrin-deficient mice are sufficient to induce thrombotic events in hematopoietically ablated recipients.

Thrombotic events are life-threatening complications of human hemolytic anemias such as paroxysmal nocturnal hemoglobinuria, sickle cell disease, and thalassemia. It is not clear whether these events are solely influenced by aberrant hematopoietic cells or also involve aberrant nonhematopoietic cells. Spherocytosis mutant (Spna1(sph)/Spna1(sph); for simplicity referred to as sph/sph) mice develop a severe hemolytic anemia postnatally due to deficiencies in -spectrin in erythroid and other as yet incompletely defined nonerythroid tissues. Thrombotic lesions occur in all adult sph/sph mice, thus providing a hematopoietically stressed model in which to assess putative causes of thrombus formation. To determine whether hematopoietic cells from sph/sph mice are sufficient to initiate thrombi, bone marrow from sph/sph or +/+ mice was transplanted into mice with no hemolytic anemia. One set of recipients was lethally irradiated; the other set was genetically stem cell deficient. All mice implanted with sph/sph marrow, but not +/+ marrow, developed severe anemia and histopathology typical of sph/sph mice. Histological analyses of marrow recipients showed that thrombi were present in the recipients of sph/sph marrow, but not +/+ marrow. The results indicate that the -spectrin-deficient hematopoietic cells of sph/sph mice are the primary causative agents of the thrombotic events.

Identification of a stable semiquinone intermediate in the purified and membrane bound ubiquinol oxidase-cytochrome bd from Escherichia coli.

The quinol oxidase, cytochrome bd, functions as a terminal oxidase in the Escherichia coli respiratory chain, reducing O2 to water and using ubiquinol-8 or menaquinol-8 as its immediate reductant. The oxidation of quinol is by the low-spin ferri-haem, cytochrome b558. This occurs at a quinol-binding site by sequential one electron steps, requiring the stabilisation of the semiquinone intermediate. We have observed, by EPR spectroscopy, the properties of this semiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess of ubiquinone-8 and menaquinone-8 analogues. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a semiquinone anion of this type. The line shape is Gaussian. The semiquinone is highly stabilised with respect to free semiquinone; significant free radical can be observed at pH 7.0 and above. The pH dependence of the redox reactions indicate that the anionic form of the semiquinone and the neutral form of the quinol predominate in the pH range studied. The pH dependence of the mid-point potentials of the one electron reactions from pH 7.0-9.0 is 120 mV/pH change for the semiquinone anion to quinol (E2) and none for the quinone to semiquinone (E1). The semiquinone radical is attenuated on titration with putative inhibitors of this quinone-binding site. We have similarly studied the semiquinone in membrane preparations from a strain with overexpression of cytochrome bd oxidase. The data can be fitted with the assumption of a single quinone-binding site.

Thrombosis and secondary hemochromatosis play major roles in the pathogenesis of jaundiced and spherocytic mice, murine models for hereditary spherocytosis.

Jaundiced mice, ja/ja, suffer from a severe hemolytic anemia caused by a complete deficiency of erythroid beta-spectrin. We used these mice as a model to investigate the pathophysiological consequences of the deficiency, including the effects in the nonerythroid tissues where this protein is expressed. Because the ja/ja mice rarely survive beyond the fourth postnatal day, methods were assessed for extending lifespan into adulthood. Neonatal transfusion increased lifespan to a mean of 3.7 months, allowing a more complete characterization of the pathophysiology. Blood parameters and histopathology of the jaundiced mouse were compared with that from spherocytic mice, which have a hemolytic anemia caused by deficiency of erythroid alpha-spectrin, yet can survive the postnatal period transfusion free. The adult jaundiced and spherocytic mice present with greatly decreased hematocrit and red blood cell counts, reticulocytosis, and bilirubinemia, leading secondarily to hepatosplenomegaly and cardiomegaly. Jaundiced and spherocytic mice were analyzed histopathologically between 1.0 and 9.5 months of age. Interestingly, the complete absence of erythroid beta-spectrin in jaundiced mice leads to no detectable structural defects in brain, cardiac, or skeletal muscles. However, fibrotic lesions and lymphocytic infiltration were observed in cardiac tissue from 4 of 13 jaundiced mice and 15 of 15 spherocytic mice, and thrombi were detected at either the atrioventricular valves or within the atria of 2 of 13 jaundiced mice and 15 of 15 spherocytic mice. In addition, all affected mice had a progressive renal hemosiderosis concurrent with hydronephrosis and glomerulonephritis. The severity of the renal disease and its presence in all moribund mice suggests kidney failure rather than the fibrotic heart lesions as the major cause of death in these mice.

Resonance Raman spectroscopic identification of a histidine ligand of b595 and the nature of the ligation of chlorin d in the fully reduced Escherichia coli cytochrome bd oxidase.

Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d3+ to yield entirely the high-spin form of the cofactor.

Methionine-393 is an axial ligand of the heme b558 component of the cytochrome bd ubiquinol oxidase from Escherichia coli.

The cytochrome bd oxidase is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. The complex is composed of two subunits (I and II) and three heme prosthetic groups (heme b558, heme b595, and a chlorin, called heme d). Both subunits are located within the bacterial cytoplasmic membrane, and each has multiple putative transmembrane helices. Heme b558 is a six-coordinate, low-spin heme component of the oxidase which has been shown to be contained within subunit I and has been implicated in the oxidation of the substrate, ubiquinol-8, in the cytoplasmic membrane. Previous site-directed mutagenesis studies identified His186, predicted to be near the periplasmic side of transmembrane helix D of subunit I, as one of the axial ligands of heme b558. Since mutagenesis of none of the other histidines in subunit I perturbs heme b558, it was concluded that this heme cannot have bis(histidine) ligation. In this work, the properties of 14 mutants are reported, including substitutions for each of 10 methionine residues within subunit I. Among this set of mutants, only the replacement of M393 perturbs heme b558. Replacement of M393 by leucine results in the conversion of heme b558 to a high-spin state. Surprisingly, the M393L mutation does not eliminate enzymatic activity, and the mutant oxidase has sufficient turnover to support aerobic growth of the cells. The addition of imidazole to the purified M393L oxidase converts heme b558 back to a low-spin configuration. The data strongly suggest that the sixth axial ligand of heme b558 is methionine-393, and that this heme, therefore, has histidine-methionine ligation. The results are consistent with recent cryogenic near-infrared magnetic circular dichroism spectra that also indicate histidine-methionine ligation of heme b558.

Resonance Raman studies of Escherichia coli cytochrome bd oxidase. Selective enhancement of the three heme chromophores of the "as-isolated" enzyme and characterization of the cyanide adduct.

Cytochrome bd oxidase is a terminal bacterial oxidase containing three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be at a dinuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinone. One of the unique functional features of this enzyme is its resistance to high concentrations of cyanide (Ki in the millimolar range). With the appropriate selection of laser lines, the ligation and spin states of the b558, b595, and d hemes can be probed selectively by resonance Raman (rR) spectroscopy. Wavelengths between 400 and 500 nm predominantly excite the rR spectra of the b558 and b595 chromophores. Spectra obtained within this interval show a mixed population of spin and ligation states arising from b558 and b595, with the former more strongly enhanced at higher energy. Red excitation wavelengths (590-650 nm) generate rR spectra characteristic of chlorins, indicating the selective enhancement of the d heme. These rR results reveal that cytochrome bd oxidase "as isolated" contains the b558 heme in a six-coordinate low-spin ferric state, the b595 heme in a five-coordinate high-spin (5cHS) ferric state, and the d heme in a mixture of oxygenated (FeIIO2 <--> FeIIIO2-; d650) and ferryl-oxo (FeIV = O; d680) states. However, the rR spectra of these two chlorin species indicate that they are both in the 5cHS state, suggesting that the d heme is lacking a strongly coordinated sixth ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

The haem b558 component of the cytochrome bd quinol oxidase complex from Escherichia coli has histidine-methionine axial ligation.

The cytochrome bd ubiquinol oxidase from Escherichia coli is induced when the bacteria are cultured under microaerophilic or low-aeration conditions. This membrane-bound respiratory oxidase catalyses the two-electron oxidation of ubiquinol and the four-electron reduction of dioxygen to water. The oxidase contains three haem prosthetic groups: haem b558, haem b595 and haem d. Haem d is the oxygen binding site, and it is likely that haem d and b595 form a bimetallic site in the enzyme. Haem b558 has been previously characterized spectroscopically as being low spin and has been shown to be located within subunit I (CydA) of this two-subunit enzyme. It is likely that haem b558 is associated with the quinol oxidation site, which has also been shown to be within subunit I. In a previous effort to locate the specific amino acids axially ligated to haem b558, all six histidines within subunit I were altered by site-directed mutagenesis. Only one, histidine-186, was identified as a likely ligand to haem b558. Hence it was suggested that haem b558 could not have bis(histidine) ligation. In the current work, a combination of low-temperature near-infrared magnetic circular dichroism (NIR-MCD) and EPR spectroscopies have been employed to identify the nature of the haem b558 axial ligands. The NIR-MCD spectrum at cryogenic temperatures is dominated by the low-spin haem b558 component of the complex, and the low-energy band near 1800 nm is strong evidence for histidine-methionine ligation. It is concluded that haem b558 is ligated to histidine-186 plus one of the methionines located within subunit I of the oxidase.

The murine mutation jaundiced is caused by replacement of an arginine with a stop codon in the mRNA encoding the ninth repeat of beta-spectrin.

The jaundiced, ja/ja, mouse mutant has a severe hemolytic anemia associated with a deficiency of beta-spectrin in erythrocyte ghosts. Genes for the disease phenotype and beta-spectrin colocalize on Chromosome 12. beta-Spectrin mRNA is not detected in reticulocytes or in brain from newborn mutant mice. To locate the nucleotide sequence alteration, the erythroid beta-spectrin transcript from mutant spleen was amplified by reverse transcription PCR and sequenced. A C-to-T alteration is present in the mutant transcript and produces a premature stop codon from an arginine codon in mRNA encoding repeat 9 of beta-spectrin at amino acid position 1160. The point mutation introduces a Dde I site that is present in PCR-amplified DNA of ja/ja and ja/+ mice but not of +/+ control mice from the strain of origin, 129/Sv, or from the two strains, WB/Re and C57BL/6J, in which the mutation has been fixed by over 53 generations of backcrossing. The genetic data confirm that the point mutation is responsible for the severe reductions in beta-spectrin mRNA of jaundiced mice.

Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii: comparison to the complex from Escherichia coli.

Partial purification of a cytochrome bd complex from Azotobacter vinelandii grown under high aeration was achieved by isolating respiratory particles enriched in this hemoprotein via differential centrifugation and detergent extraction. The cytochrome bd complex was subsequently solubilized from the inner membrane with dodecyl maltoside and purified to near homogeneity via DEAE-Sepharose chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the complex consisted of two subunits, with sizes in good agreement with those predicted from the cloned cyd locus (59.7 and 42 kDa). Spectral analysis of the purified complex indicated that the heme components present were cytochromes b560, b595, and d; CO difference spectral studies identified cytochrome d as a CO-reactive component. The complex had a Km for ubiquinol-1 approximately seven times larger than that for the analogous bd complex from Escherichia coli, and O2 consumption curves revealed a Km value for O2 three times greater than that which we determined for the E. coli bd complex.