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.     

Regulation of mitochondrial morphology and cell survival by Mitogenin I and mitochondrial single-stranded DNA binding protein

We found that a mouse homolog of human DNA polymerase delta interacting protein 38, referred to as Mitogenin I in this paper, and mitochondrial single-stranded DNA-binding protein (mtSSB), identified as upregulated genes in the heart of mice with juvenile visceral steatosis, play a role in the regulation of mitochondrial morphology. We demonstrated that overexpression of Mitogenin I or mtSSB increased elongated or fragmented mitochondria in mouse C2C12 myoblast cells, respectively. On the other hand, the silencing of Mitogenin I or mtSSB by RNA interference led to an increase in fragmented or elongated mitochondria in the cells, respectively, suggesting that Mitogenin I and mtSSB are involved in the processes of mitochondrial fusion and fission, respectively. In addition, we showed that the silencing of Mitogenin I resulted in an increase in the number of trypan blue-positive cells and the silencing of mtSSB resulted in an enhancement of the sensitivity of the cells to apoptotic stimulation by etoposide. The present results demonstrated that these proteins play a role in cell survival.     

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.     

Enzyme kinetic and spectroscopic studies of inhibitor and effector interactions with indoleamine 2,3-dioxygenase. 2. Evidence for the existence of another binding site in the enzyme for indole derivative effectors

To probe the active site of the heme protein indoleamine 2,3-dioxygenase, the effects of 3-indoleethanol (IET) (or tryptophol), one of the known indole derivative effectors, and indole (IND) on the catalytic (Vmax, Km) and spectroscopic properties (optical absorption and CD) of the enzyme were investigated. Assays were performed with the substrate L- or D-tryptophan (Trp) and an ascorbic acid-methylene blue cofactor system at 25 degrees C. This study has shown that, at millimolar concentrations, both IET and IND lower considerably the Km value for D-Trp by approximately 25% and approximately 60%, respectively, at pH 7.0, while neither affects the Km value for L-Trp. Interestingly, however, these effectors exert opposite effects with respect to each other on the Vmax values for both D-Trp and L-Trp: IET enhances the Vmax values by 40-60% while IND lowers them by 12-24%. These effects of IET and IND on the Vmax values may be attributed to the shift in the ferric (inactive) enzyme----ferrous (active) enzyme equilibrium either to the right (IET) or to the left (IND) caused by the binding of these effectors to the enzyme in the steady state of the catalytic reaction. Both effectors induce clearly detectable spectral changes, especially notable in CD spectra, upon binding (in a 1:1 molar ratio, Kd = 10(-4) to 2.5 X 10(-3) M) to the ferrous enzyme and its complexes with O2, CO, and NO, both in the presence and in the absence of L-Trp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic and equilibrium properties of the indoleamine 2,3-dioxygenase-tryptophan-O2 ternary complex and of analogous enzyme derivatives. Tryptophan binding to ferrous enzyme adducts with dioxygen, nitric oxide, and carbon monoxide

The dioxygen adduct of the heme protein indoleamine 2,3-dioxygenase has been generated at -30 degrees C in mixed solvents, and spectroscopic and equilibrium studies of its L-tryptophan (substrate) binding properties have been carried out for the first time. Comparative studies have also been performed with the NO and CO adducts of the ferrous enzyme. Under the conditions employed (-30 degrees C), both autoxidation and turnover (L-tryptophan + O2----formylkynurenine) of the ternary complex are effectively suppressed. Structural identification of the ternary complex is based on the 1:1 molar stoichiometry for the substrate-oxygenated enzyme adduct formation (Kd approximately 10(-4) M), the time-dependent linear product formation (turnover) at -20 degrees C, and the quantitative conversion of the complex to the ferrous CO derivative by bubbling with CO. Binding of L-tryptophan to the oxygenated enzyme leads to decreases in the intensities of its major absorption bands (lambda max 415, 541, 576 nm) and to a blue shift of its Soret peak. Interestingly, among the ferrous enzyme derivatives examined, only the substrate-bound oxygenated enzyme exhibits solvent-dependent Soret absorption peak positions, e.g., lambda max 411.5 and 413.5 nm in 65% (v/v) aqueous glycerol and ethylene glycol, respectively. In addition, indole binds to the oxygenated enzyme, causing a red shift of its Soret peak in these solvents only in the presence of substrate (411.5----414 nm and 413.5----414.5 nm, respectively), while similar effects of indole are independent of tryptophan for the other ferrous enzyme derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.