Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 1. Siroheme vibrational modes

Resonance Raman (RR) spectra are reported for the hemoprotein subunit (SiR-HP) of Escherichia coli NADPH-sulfite reductase (EC 1.8.1.2) in various ligation and redox states. Comparison of the RR spectra of extracted siroheme and the mu-oxo FeIII dimer of octaethylisobacteriochlorin with those of mu-oxo FeIII octaethylchlorin dimer and mu-oxo FeIII octaethylporphyrin dimer demonstrates that many siroheme bands can be correlated with established porphyrin skeletal modes. Depolarization measurements are a powerful tool in this correlation, since the 45 degrees rotation of the C2 symmetry axis of the isobacteriochlorin ring relative to the chlorin system results in reversal of the polarization properties (polarized vs anomalously polarized) of bands correlating with B1g and B2g modes of porphyrin. Various SiR-HP adducts (CO, NO, CN-, SO3(2-] show upshifted high-frequency bands, characteristic of the low-spin state and consistent with the expected core size sensitivity of the skeletal modes. Fully reduced unliganded SiR-HP (both siroheme and Fe4S4 cluster reduced) in liquid solution displays RR features comparable to those of high-spin ferrous porphyrins; on freezing, the RR spectrum changes, reflecting an apparent mixture of siroheme spin states. At intermediate reduction levels in solution a RR species is observed whose high-frequency bands are upshifted relative to oxidized and fully reduced SiR-HP. This spectrum, thought to arise from the "one-electron" state of SiR-HP (siroheme reduced, cluster oxidized), may be due to S = 1 FeII siroheme.     

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 3. Bound ligand vibrational modes

The vibrations of the bound diatomic heme ligands CO, CN-, and NO are investigated by resonance Raman spectroscopy in various redox states of Escherichia coli sulfite reductase hemoprotein, and assignments are generated by use of isotopically labeled ligands. For the fully reduced CO complex (ferrous siroheme, reduced Fe4S4 cluster) at room temperature, nu CO is observed at 1904 cm-1, shifting to 1920 cm-1 upon oxidation of the cluster. The corresponding delta FeCO modes are identified at 574 and 566 cm-1, respectively, by virtue of the zigzag pattern of their isotopic shifts. In frozen solution, two species are observed for the cluster-oxidized state, with nu CO at 1910 and 1936 cm-1 and nu FeC at 532 and 504 cm-1, respectively; nu FeC for the fully reduced species is identified at 526 cm-1 in the frozen state. For the ferrous siroheme-NO complex (cluster oxidized), nu NO is identified at 1555 cm-1 in frozen solution and a low-frequency mode is identified at 558 cm-1; this stretching mode is significantly lower than that observed in Mb-NO. For the ferric siroheme cyanide complexes evidence of two ligand-bonding forms is observed, with modes at 451/390 and 451/352 cm-1; they are distinguished by a reversal of the isotopic shift patterns of the upper and lower modes and could arise from a linear and a bent Fe-C unit, respectively. For the ferrous siroheme cyanide complex isotope-sensitive modes observed at 495 and 452 cm-1 are assigned to the FeCN- bending and FeC stretching vibrations, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Post-translational addition of chondroitin sulfate glycosaminoglycans. Role of N-linked oligosaccharide addition, trimming, and processing

A melanoma proteoglycan model system has been used to examine the role of core protein asparagine-linked (N-linked) oligosaccharides in the transport and assembly of proteoglycan molecules. The use of agents which block discrete steps in the trimming and processing of core oligosaccharides (castanospermine, 1-deoxynojirimycin, N-methyldeoxynojirimycin, 1-deoxymannojirimycin, and swainsonine) demonstrates that removal of glucose residues from the N-linked oligosaccharides is required for the cell surface expression of a melanoma proteoglycan core protein and for the conversion of the core protein to a chondroitin sulfate proteoglycan. However, complete maturation of the oligosaccharides to a "complex" form is not required for these events. Treatment of M21 human melanoma cells with the glucosidase inhibitors castanospermine, 1-deoxynojirimycin, or N-methyldeoxynojirimycin results in a dose-dependent inhibition of glycosaminoglycan (GAG) addition to the melanoma antigen recognized by monoclonal antibody 9.2.27. In contrast, treatment with the mannosidase inhibitors 1-deoxymannojirimycin and swainsonine does not effect GAG addition. Identical results are obtained when the major histocompatibility complex class II antigen gamma chain proteoglycan is examined in inhibitor-treated melanoma and B-lymphoblastoid cells. These data, in conjunction with the known effects of the glucosidase and mannosidase inhibitors on the transport and secretion of other glycoproteins support the hypothesis that the addition, trimming, and processing of N-linked oligosaccharides is involved in the transport of certain proteoglycan core proteins to the site of GAG addition and to the cell surface.     

Presence of sulfate in N-glycosidically linked carbohydrate units of calf thyroid plasma membrane glycoproteins

Calf thyroid slices were found to incorporate [35S] sulfate into two major plasma membrane glycoproteins, which have been previously designated as GP-1 and GP-3 (Okada, Y., and Spiro, R. G. (1980) J. Biol. Chem. 255, 8865-8872). The 35S-glycoproteins were identified on the basis of their characteristic solubility and electrophoretic migration as well as their affinity for Bandeiraea simplicifolia I lectin. After pronase digestion of these glycoproteins, the 35S-label remained associated with the glycopeptides primarily on asparagine-linked carbohydrate units which were released by hydrazinolysis. Examination of the reduced radio-labeled products obtained by nitrous acid cleavage of the hydrazine-liberated oligosaccharides indicated that sulfate esters of N-acetylglucosamine occurred at three locations on the carbohydrate units; two 35S-monosaccharides (2,5-anhydromannitol 4- and 6-sulfate) and one 35S-disaccharide (beta-Gal(1----4)-2,5-anhydromannitol(6-SO4] were formed. The disaccharide is believed to be derived from an internal sulfated N-acetyllactosamine sequence while the monosaccharides most likely originate from 4- and 6-sulfated N-acetylglucosamine residues situated, respectively, at the non-reducing and reducing termini of the oligosaccharide units. Quantitation by NaB[3H]4 reduction of the sulfated saccharides obtained by nitrous acid treatment of hydrazine-released oligosaccharides from unlabeled GP-3 indicated that about 20% of the asparagine-linked carbohydrate units contain sulfate substituents.     

Dynamics of carbon monoxide binding to cystathionine beta-synthase

Cystathionine beta-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 microm(-1). Stopped flow measurements reveal slow CO association (0.0166 s(-1)) limited by dissociation of the endogenous ligand, Cys-52. Rebinding of CO and of Cys-52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys-52 rebinding rate, 4000 s(-1), is essentially unchanged between pH 7.6 and 10.5, indicating that the pK(a) of Cys-52 is shifted below pH 7.6. This effect is attributed to the nearby Arg-266 residue, which is proposed to form a salt bridge with the dissociated Cys-52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg-266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.