Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy

In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.     

Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin.

Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins.     

Kinetic and equilibrium studies of the reactions of heme-substituted horse heart myoglobins with oxygen and carbon monoxide.

In order to study the effects of chemical modifications of the vinyl groups of heme on oxygen and carbon monoxide binding to myoglobin, apomyoglobins from horse heart were reconstituted with six different hemins with various side chains. Laser flash photolysis experiments of these reconstituted myoglobins showed that the combination rate constants for oxygen (k') and carbon monoxide (l') were closely related to the electron-attractive properties of the side chains. The k' values obtained in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees were 0.83 (meso-), 2.4 (deutero-), 1.1 (reconstituted proto-), 1.2 (native proto-), 1.5 (2-formyl-4-vinyl-), 1.9 (2-vinyl-4-formyl-), and 2.7 X 10(7) M-1 S-1 (2,4-diformylmyoglobins), and the corresponding l' values were 2.8, 18, 4.8, 5.1, 7.1, 15, and 35 X 10(5) M-1 S-1, respectively. These rate constants tend to increase as the electron-withdrawing power of the side chains increases, indicating that reduced electron density of the iron atom of heme in myoglobin favors the combination reaction for both oxygen and carbon monoxide. Equilibrium constants (L) between carbon monoxide and various myoglobins were also determined by measuring the partition coefficients (M) between oxygen and carbon monoxide for the myoglobins, and were also found to be closely related to the electronic properties (pK3 of porphyrin) of the heme side chains. The equilibrium association constants for carbon monoxide thus obtained increased with a decrease in pK3 value of the porphyrin. This order was completely opposite to the case of the oxygen binding reaction. The dissociation rate constants for oxygen (k) and carbon monoxide (l) were calculated from the equilibrium and the combination rate constants. The dissociation rate constants showed a similar characteristic to the combination rate constants and increased with the increase in electron attractivity of heme side chains. The concomitant increase in both the combination and dissociation rate constants with increase in electronegativity of the iron atom suggests that these reactions have different rate determining steps, although such a reaction process is contradictory to the generally accepted concept that in a reversible reaction, both on and off reactions proceed through the same transition state. In the on reaction sigma bond formation appears to be dominant, while in the off reaction eta bond break-up is more important.     

Decrease in oxygen affinity of myoglobin by formylation of vinyl groups of heme.

Three kinds of green synthetic myoglobin were prepared by recombination of horse heart apomyoglobin with spirographis (2-formyl-4-vinyl-), isospirographis (2-vinyl-4-formyl-), and 2,4-diformyldeuterohemins. The optical and oxygen binding properties of the reconstituted myoglobins containing two isomeric monoformyl-monovinylhemins were found to be different. The oxygen affinities (P50) of spirographis and 2,4-diformylmyoglobins are 2.7 and 2.8 mm Hg, respectively, at 25 degrees, and about 2.5 times lower than that of native protomyglobin, while that of isospirographis myoglobin is 1.0 mm Hg and is similar to native myoglobin. Spirographis oxymyoglobin has absorption maxima (alpha, beta, and Soret bands) at 601, 556.5, and 435 nm, isospirographis oxymyoglobin at 595, 550, 429 nm, and 2,4-diformyl oxymyoglobin at 603, 563.5, and 447 nm. The optical red shifts as well as the decrease in the oxygen affinities of these myoglobins are attributed mainly to the presence of strongly electron-attractive formyl side chains. Since the free isomers of monoformyl-monovinyl heme have similar properties, the differences observed after recombination with apoprotein must be caused by interactions with apomyoglobins. The degree of such a protein effect may be estimated by comparing the absorption spectra of heme before and after recombination and was found to differ among the various myoglobins. Comparison of the oxygen affinities of the myoglobins taking account of this protein factor showed that the increase in the P50 values are inversely related to that in the pK3 values of the free porphyrins. These results suggest the involvement of pi bonding in determining the oxygen-iron bond strength.     

EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or N(G)-hydroxyarginine

Reconstitution of the endothelial nitric oxide synthase heme domain (NOS) with the catalytically noncompetent 4-aminotetrahydrobiopterin has allowed us to prepare at -40 degrees C the oxyferrous-NOS-substrate complexes of both L-arginine (Arg) and N(G)-hydroxyarginine (NOHA). We have radiolytically cryoreduced these complexes at 77 K and used EPR and ENDOR spectroscopies to characterize the initial products of reduction, as well as intermediates that arise during stepwise annealing to higher temperatures. Peroxo-ferri-NOS is the primary product of 77 K cryoreduction when either Arg or NOHA is the substrate. Proton ENDOR spectra of this state suggest that the peroxo group is H-bonded to a [guanidinium-water] network that forms because the binding of O2 to the ferroheme of NOS recruits H2O. At no stage of reaction/annealing does one observe an EPR signal from a hydroperoxo-ferri state with either substrate. Instead, peroxo-ferri-NOS-substrate complexes convert to a product-state intermediate at the extremely low temperature of 165-170 K. EPR and proton ENDOR spectra of the intermediate formed with Arg as substrate support the suggestion that the reaction involves the formation and attack of Compound I. Within the time/temperature resolution of the present experiments, samples with Arg and NOHA as substrate behave the same in the initial steps of cryoreduction/annealing, despite the different acid/base characteristics of the two substrates. This leads us to discuss the possibility that ambient-temperature catalytic conversion of both substrates is initiated by reduction of the oxy-ferroheme to the hydroperoxo-ferriheme through a coupled proton-electron transfer from a heme-pocket reductant, and that Arg may provide the stoichiometrically second proton of catalysis.     

Four alkaloids, lucidine B, oxolucidine A, lucidine A, and lucidulinone from Lycopodium lucidulum

The structures of four alkaloids extracted from Lycopodium lucidulum (Lycopodiaceae) were established by X-ray and 2D NMR spectroscopic analyses. The dihydro-derivative of oxolucidine A, which was obtained by NaBH4 reduction of oxolucidine A, was treated with p-bromobenzoyl chloride to afford crystals, whose X-ray crystallographic analysis established the stereostructure, including the absolute configuration. The 2D NMR spectra of tetrahydrodeoxylucidine B were fully analyzed to establish the full structure of lucidine B, and the hitherto unknown stereochemistry at the C-14 position was established as beta-H. The structure of a new alkaloid, lucidulinone, was determined by spectroscopic analysis to be luciduline lactam.     

Sphingolipids are required for the stable membrane association of glycosylphosphatidylinositol-anchored proteins in yeast

Ongoing sphingolipid synthesis is specifically required in vivo for the endoplasmic reticulum (ER) to Golgi transport of glycosylphosphatidylinositol (GPI)-anchored proteins. However, the sphingolipid intermediates that are required for transport nor their role(s) have been identified. Using stereoisomers of dihydrosphingosine, together with specific inhibitors and a mutant defective for sphingolipid synthesis, we now show that ceramides and/or inositol sphingolipids are indispensable for GPI-anchored protein transport. Furthermore, in the absence of sphingolipid synthesis, a significant fraction of GPI-anchored proteins is no longer associated tightly with the ER membrane. The loose membrane association is neither because of the lack of a GPI-anchor nor because of prolonged ER retention of GPI-anchored proteins. These results indicate that ceramides and/or inositol sphingolipids are required to stabilize the association of GPI-anchored proteins with membranes. They could act either by direct involvement as membrane components or as substrates for the remodeling of GPI lipid moieties.