Aditya P. Koley

DIFFERENTIAL MECHANISMS OF CYTOCHROME P450 INHIBITION AND ACTIVATION BY ALPHA -NAPHTHOFLAVONE

The anticarcinogenicity of some flavonoids has been attributed to modulation of the cytochrome P450 enzymes, which metabolize procarcinogens to their activated forms. However, the mechanism by which flavonoids inhibit some P450-mediated activities while activating others is a longstanding, intriguing question. We employed flash photolysis to measure carbon monoxide binding to P450 as a rapid kinetic technique to probe the interaction of the prototype flavonoid α-naphthoflavone with human cytochrome P450s 1A1 and 3A4, whose benzo[a]pyrene hydroxylation activities are respectively inhibited and stimulated by this compound. This flavonoid inhibited P450 1A1 binding to benzo[a]pyrene via a classical competitive mechanism. In contrast, α-naphthoflavone stimulated P450 3A4 by selectively binding and activating an otherwise inactive subpopulation of this P450 and promoting benzo[a]pyrene binding to the latter. These data indicate that flavonoids enhance activity by increasing the pool of active P450 molecules within this P450 macrosystem. Activators in other biological systems may similarly exert their effect by expanding the population of active receptor molecules.

Drug-drug interactions: Effect of quinidine on nifedipine binding to human cytochrome P450 3A4

Quinidine is a known inhibitor of cytochrome P450-mediated nifedipine metabolism. The interactions of nifedipine and quinidine with human cytochrome P450 3A4, which metabolizes these drugs, were examined using the kinetics of CO binding to this P450 as a rapid kinetic probe of protein conformation and dynamics. This approach showed that nifedipine and quinidine bind to different P450 3A4 species, respectively termed species I and II, with distinct conformations. When both drugs were present simultaneously, nifedipine interacted with the quinidine-bound P450 species II, but not species I. These findings indicate that quinidine acts as an allosteric inhibitor by switching nifedipine binding from nifedipine-metabolizing species I to the nonmetabolizing species II.

Cytochrome P450 conformation and substrate interactions as probed by CO binding kinetics.

The kinetics of CO binding to cytochrome P450, as measured by the flash photolysis technique, is a powerful probe of P450 structure-function relationships. The kinetics are sensitive to P450 conformation and dynamics and are modulated by P450 interactions with substrates and other components of the microsomal membrane. Application of a difference method to kinetic data analysis distinguishes the kinetic behavior of individual P450 forms in the microsomal membrane. This approach shows that substrates differentially modulate the kinetics via: 1) changes in P450 conformation/dynamics that either accelerate or reduce the binding rate; and/or 2) steric effects that reduce the rate. Both mechanisms are observed, the relative contributions of each varying in a substrate- and P450-dependent manner. In addition to microsomes, substrate interactions with individual P450s can be similarly probed using expressed P450s. Experiments with baculovirus-expressed human P450 3A4 show that this P450 consists of multiple conformers with distinct substrate specificities, an observation which provides a basis for its recognition of a wide array of structurally diverse substrates. These studies thus demonstrate the utility of CO binding kinetics in elucidating fundamental P450-substrate interactions in a biological membrane environment.

Modification of α-Chain or β-Chain Heme Pocket Polarity by Val(E11) → Thr Substitution Has Different Effects on the Steric, Dynamic, and Functional Properties of Human Recombinant Hemoglobin DEOXY DERIVATIVES

The dynamic and functional properties of mutant deoxyhemoglobins in which either the β-globin Val67(E11) or the α-globin Val62(E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified α chain and β chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the α chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the β chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified α chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the α chain distal heme pocket can bridge by H bonding between Thr62(E11) and His58(E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified β chains is not observed by x-ray crystallography.

Chemistry of molybdenum with hard-soft donor ligands—III. Oxomolybdenum(VI) and cis-dioxomolybdenum(VI) mixed ligand complexes

Abstract The syntheses and characterization of a number of oxomolybdenum(VI) and cis -dioxomolybdenum(VI) complexes with 4-substituted thiosemicarbazides in the form of 4-phenyl, 4-( p -methylphenyl) and 4-( p -chlorophenyl)thiosemicarbazide (represented as LH) as the NS donor metal binding substrates are described. The oxomolybdenum(VI) complexes are of the type MoO(NS) 2 X 2 and MoO(NS) 2 (OO) (where X = Br or SCN and OO = oxalate or malonate) with a pentagonal bipyramidal geometry. The cis -dioxomolybdenum(VI) complexes are mixed chelate complexes, MoO 2 (NS)L′, where L′H is either a monoprotic NO donor like oxine, 2-methyloxine or a NS donor such as 2-aminobenzenethiol, 2-aminocyclopent-1-ene-1-dithiocarboxylic acid and its methyl ester. The electrochemical behaviour of these complexes along with cis -MoO 2 (NS) 2 and MoO(NS) 2 Cl 2 complexes are discussed.

Assembly of Human Hemoglobin STUDIES WITH ESCHERICHIA COLI-EXPRESSED α-GLOBIN

The α-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native β-chains. The functional and structural properties of the expressed α-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer. The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl− and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the α1β1 and α1β2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed α-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA. These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics. In addition to incorporation into a HbA tetramer, the α-globin refolds and incorporates heme in the absence of the partner β-chain. Although the CO binding kinetics of recombinant α-chains were the same as that of native α-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component. These results show that recombinant α-chain assumes alternating conformations in the absence of β-chain and indicate that the isolated α-chain exhibits a higher degree of conformational flexibility than the α-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed α-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.

A paramagnetic octahedral trans-dihydroxy chromium(IV) complex with dianionic tetradentate Schiff base salophen and crystal structure of its trans-diisothiocyanato analog

A paramagnetic octahedral trans-dihydroxychromium(IV) complex, [Cr(OH)2(salophen)] (1) (H2salophen = N,N′-bis(salicylidene)-1,2-phenylenediamine), has been synthesized and characterized by elemental analysis, magnetic moment measurement, IR, UV-Vis, and EPR spectroscopic studies. Measured room temperature (RT) magnetic moment value is 2.79 BM for 1, indicating a d2 (S = 1) system with a triplet ground state. Compound 1 exhibits powder EPR spectra at RT and LNT, which show the allowed transition ΔM s = ±1 (g = 2.0038) as well as the “forbidden” half-field transition (ΔM s = ±2) at g = 4.2080. Two successive reduction waves are observed in the cyclic voltammogram of 1 in CH3CN at −0.84 and −1.63 V (vs. Ag/AgCl), respectively. Compound 1 readily reacts with Mn2+ ion, a Cr(IV)–specific reductant and also undergoes –OH substitution reactions in solution with NCS− and imidazole. The trans-diisothiocyanato analog, [Cr(NCS)2(salophen)] (2), with μ eff = 2.80 BM has been structurally characterized by X-ray crystallography and found to contain two N-bonded axial thiocyanato ligands with slightly different axial Cr–N bond lengths (N(3)–Cr(1), 2.032(2); N(4)–Cr(1), 2.015(2) Å). Compound 2 and the corresponding Cr(III) compound K[Cr(NCS)2(salophen)] · H2O (3) show significant difference in their electronic structures as revealed from their electronic spectra.

First direct detection of chromium(IV) as a long lived intermediate in the oxidation of methanol by chromium(VI)

Reduction of chromium(VI) by excess methanol in solutions buffered by 2-ethyl-2-hydroxy butanoic acid and its sodium salt proceeds through a 2-electron path to produce pink chromium(IV). With the progress of this reaction, a slow oxidation of the product chromium(IV) by reactant chromium(VI) takes place. When all the chromium(VI) is consumed, the chromium(IV) undergoes very slow disproportionation to chromium(V) and chromium(III). The formation of chromium(IV) is accelerated by both 2-ethyl-2-hydroxy butanoate ion and hydrogen ion.

Dioxygen binding and activation by a highly reactive Cr(II) compound containing S,N-donors derived from o-aminothiophenol

We report the synthesis, characterization, and reactivity of a Cr(II) complex, [Cr(H2O)(LISQ)2] (1) [(LISQ)1− is o-iminothionebenzosemiquinonate(1−) π-radical], that is highly stable in solid state in the presence of air but undergoes spontaneous change in solution, both in the presence and absence of air. Physicochemical studies in solution show that a superoxo-CrIII species, [Cr(O2)(OH)(LISQ)2]− is generated initially in DMF solution of 1 in the presence of air owing to its immediate deprotonation followed by O2 binding to the deprotonated species. The formation of this superoxo-CrIII species is prominent and gradual in the presence of CH3OH, a scavenger of CrO2+ species. This Cr(O2)2+ species in turn is converted to another highly reactive O=Cr(IV) intermediate [O=Cr(OH)(LISQ)2]− which undergoes disproportionation producing an unstable O=Cr(V) species, [O=Cr(OH)(LISQ)2] and a stable Cr(III) compound, [Cr(OH)(DMF)(LISQ)2] (2). The rate of this disproportionation is enhanced in the presence of MnCl2, [N(n-Bu)4]PF6 and KSCN. The generated O=Cr(IV) species interacts with DNA with complete cleavage. The O=Cr(V) species slowly disappears from solution as revealed from EPR studies.

Studies on gold(II) complexes with hard and soft donor ligands. Part I. Complexes with o-aminobenzenethiol

The synthesis and characterisation of gold(II) complexes with o-aminobenzenethiol is reported. The e.s.r. results indicate that the highest occupied molecular orbital has appreciable contribution from the sulphur donors of the ligands. It is the first time that binuclear gold(II) complexes giving well resolved seven-line e.s.r. patterns originating from two 197Au nuclei have been reported.