Heme-bound tyrosine vibrations in hemoglobin M: Resonance Raman,crystallography, and DFT calculation

Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or β subunit contains a ferric heme in the α₂β₂ tetramer. Though the ferric subunit cannot bind O₂, it regulates O₂ affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His → tyrosine [Tyr]) and M Boston (α58His → Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm^-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe³⁺-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.     

Structural nonequivalence of the alpha- and beta- heme-pockets in human methemoglobin. A proton magnetic relaxation study in solution.

Solvent-proton longitudinal magnetic relaxation rates as dependent on temperature were measured for human (H)/canine (C) valency hybrids of the type {α^H(III)β^C(II)}₂ and {α^C(II)β^H(III)}₂. The two metheme irons in the human methemoglobin chains induce quite different proton magnetic relaxation (pmr) rates reflecting a tighter β-heme-pocket compared to the α subunit. Both heme-pockets appear to be loosened in the presence of inositol hexaphosphate (IHP) although this allosteric effector binds only to the β chains, the binding assumed to be the same for canine as for human hemoglobin. The subunit nonequivalence is retained also in the T-quaternary state induced by IHP. In the species hybrids the pmr rates due to the metheme iron are sensitive to the valency (ligand) state, which was either CO or H₂O in the partner half of the hybrid. All results show very clearly the interrelationship of the tertiary (protomer) structure with the quaternary (oligomer) structure in hemoglobin.     

A simple method for preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

Reciprocal effects of change of subunit structure on ligand equilibria of haemoglobin valency hybrids. Attempted correlation with electron paramagnetic resonance spectra

Haemoglobin valency hybrids have been further investigated with a view to evaluating evidence for α-β interactions. Comparison of equilibrium oxygen binding of the compounds α^III_H2Oβ^II, α^III_Fβ^II, α^III_N3β^II and α^III_CNβ^II show that the derivatives possessing the α chain in the (low spin) oxy conformation possess higher oxygen affinity than those possessing the same chain in (high spin) deoxy conformation. On the other hand, equilibrium titration of the aquo derivative by fluoride and azide showed a higher azide affinity and a slightly lower fluoride affinity for α^III_H2Oβ^II_CO compared to the deoxy form α^III_H2Oβ^II. Essentially the same pattern of relations were obtained for the different forms of α^IIβ^II also.Electron paramagnetic resonance spectra of the hybrids at pH 6 showed no change of the g value of 5.85 absorption of the ferri chain on change of spin state of the partner ferro chain. However, this was no longer the case at pH 9; the spectra of the alkaline form gave evidence of α-β interaction for the α^IIIβ^II hybrid only, but not for α^IIβ^III. The electron paramagnetic resonance results suggest that α-β interaction in the hybrids may operate in the β → α direction only.The equilibrium and spectroscopic data are discussed in the general context of haem-haem interaction.     

Oxygen equilibria of hemoglobin A and hemoglobin S valency hybrids.

Oxygen equilibrium determinations with “unsymmetrical” MetHb/Hb hybrids derived from human hemoglobins A and S are reported. All four of the possible hybrids have higher oxygen affinity than the parent hemoglobins. The α₂^Metβ₂^S hybrid has a lower oxygen affinity than that of α₂^Metβ₂^S. However, both the β^Met hybrids have similar oxygen affinity. The Bohr value of α₂^Metβ₂^S is more negative than that of α₂^Metβ₂^A while the β^Met hybrids appear to have almost identical Bohr values. These findings favor the view that α and β chains in hemoglobin A have different conformations and indicate that hemoglobin S has a β-chain conformation different from that of β-chain of hemoglobin A. This difference is probably carried into the oxygenation properties of the α-chain in such a way as to be reflected only when the β chain is oxidized.     

Electrochemical oxidation of Mo(CO)4(LL) and Mo(CO)3(LL)(CH3CN): Generation, infrared characterization, and reactivity of [Mo(CO)4(LL)] and [Mo(CO)3(LL)(CH3CN)] (LL = 2,2′-bipyridine, 1,10-phenanthroline and related ligands)

Mo(CO)₄(LL) complexes, where LL = polypyridyl ligands such as 2,2′-bipyridine and 1,10-phenanthroline, undergo quasi-reversible, one-electron oxidations in methylene chloride yielding the corresponding radical cations, [Mo(CO)₄(LL)]⁺. These electrogenerated species undergo rapid ligand substitution in the presence of acetonitrile, yielding [Mo(CO)₃(LL)(CH₃CN)]⁺; rate constants for these substitutions were measured using chronocoulometry and were found to be influenced by the steric and electronic properties of the polypyridyl ligands. [Mo(CO)₃(LL)(CH₃CN)]⁺ radical cations, which could also be generated by reversible oxidation of Mo(CO)₃(LL)(CH₃CN) in acetonitrile, can be irreversibly oxidized yielding [Mo(CO)₃(LL)(CH₃CN)₂]²⁺ after coordination by an additional acetonitrile. Infrared spectroelectrochemical experiments indicate the radical cations undergo ligand-induced net disproportionations that follow first-order kinetics in acetonitrile, ultimately yielding the corresponding Mo(CO)₄(LL) and [Mo(CO)₂(LL)(CH₃CN)₃]²⁺ species. Rate constants for the net disproportionation of [Mo(CO)₃(LL)(CH₃CN)]⁺ and the carbonyl substitution reaction of [Mo(CO)₃(LL)(CH₃CN)₂]²⁺ were measured. Thin-layer bulk oxidation studies also provided infrared characterization data of [Mo(CO)₄(ncp)]⁺ (ncp = neocuproine), [Mo(CO)₃(LL)(CH₃CN)]⁺, [Mo(CO)₃(LL)(CH₃CN)₂]²⁺ and [Mo(CO)₂(LL)(CH₃CN)₃]²⁺ complexes.     

A simple method for preparation of valency hybrid hemoglobins, (αβ)2 and (αβ)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

Systematics and the properties of the lanthanides. : Edited by S. P. Sinha. D. Reidel,Dordrecht/Boston/Lancaster, 1983, 648 pp, 195 D fl.

The Δ-α-and Λ-β- forms of [Co(edda)CO₃]^? have been isolated as crystalline diastereoisomeric salts of the cation Δ-[Co(en)₂(ox)]⁺. When each salt is dissolved in 1.0 F aqueous Na₂CO₃ at 25°C an isomeric equilibration process occurs between these species in which the Δ-α form predominates before the eventual racemisation of both anionic species. On proceeding to equilibrium the Λ-β-[Co(edda) CO₃]^? isomer is observed to invert its absolute configuration at the central metal ion in converting to the Δ-α stereoisomer. This is the first reported example of inversion-isomerisation involving the β → α transformation in a chiral complex containing a linear tetradentate.     

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH³ and the nicotinamide ring of αNAD⁺ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD⁺ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD⁺.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10^?3 min^?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M₄ and H₄ lactate dehydrogenase, are shown to oxidize αNADH to αNAD⁺ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD⁺in vivo.     

The redox properties of (1,6-bis(benzimidazol-2-yl)-2,5-dithiahexane) chloro-copper(II) chloride as studied by cyclic voltammetry

Cyclic voltammetry in acetonitrile has been used to study the redox properties of the compound [Cu-(BBDH)Cl] Cl under various conditions. The nature of the species in solution was studied with the aid of ligand-field spectra and ESR spectra. It appears that in CH₃CN solution two Cu(II) species are predominating, viz. [Cu(BBDH)(CH₃CN)_x]²⁺ and Cu(BBDH)-Cl⁺. Minor species are dimeric in nature, such as [(BBDH)CuCl₂Cu(BBDH)]²⁺, as easily seen from ESR. The relative amount of the two major species was varied using added LiCl, allowing us to determine the nature of both species. The redox potential of the species [Cu(BBDH)Cl]⁺ appeared to be 0.62 V (against a normal hydrogen electrode), which is very high for a Cu(II) compound and in the same area as found for the blue copper proteins. Re-oxidation of Cu(BBDH)⁺ in the presence of LiCl shows that C$\text{l}$ slowly recoordinates after reoxidation.     

Conformational requirements for the polymerization of hemoglobin S: studies of mixed liganded hybrids

Gelation experiments with artificially formed half-liganded hybrid tetramers of hemoglobin S demonstrate that when either the α chains or the β^s chains are fixed in the cyanmet (CNmet) liganded state, gelation occurs upon deoxygenation of the ferrous chains. The minimum concentration of hemoglobin required for gelation is equivalent for both hybrids (α₂^cnmetβ₂^s and α₂β₂^scnmet), is considerably higher than the concentration required to gel deoxy-Hb S (α₂β₂^s), and can be restored to the lower minimum gelling point of α₂β₂^s by reduction of the CNmet chains with dithionite. These results suggest that the most important conformational determinant of the deoxy state for polymerization of Hb S is the quaternary deoxy structure rather than the tertiary structural effect of the ligand state of the α or the β^s chains, and are furthermore consistent with the notion that asymmetric deoxy-CNmet hybrid tetramers assume a conformation which resembles, but is not identical to that of deoxyhemoglobin.The results of gelation experiments with mixtures of hemoglobins S and A in which selected chains of one or both hemoglobins are in the CNmet form support the concept that certain non-S hemoglobins may participate in the sickling process by forming hybrid tetramers with Hb S (such as α₂β^aβ^s). The conformational requirement for participation of these hybrids in polymers also appears to be a quaternary deoxy-like structure.     

Observation of allosteric transition in hemoglobin

Two conclusions have been drawn from NMR studies of mixed state hemoglobins. First the α and β subunits in hemoglobin are not equivalent in their conformational properties. Second the mixed state hemoglobin (α^IIICN β^II)₂ can take two different quaternary structures without changing the degree of ligation. One of the two structures is similar to that of deoxyhemoglobin and the other to that of oxyhemoglobin.     

Specific cation effect on quenching reactions of excited tris(α,α′-diimine) ruthenium(II) and chromium(III) complexes by cyanide complexes in aqueous solutions

Specific salt effects were studied on the quenching reaction of excited [Ru(NN)₃]²⁺ (NN=2,2′-bipyridine(bpy), 1,10-phenanthrorine(phen)) and [Cr(bpy)₃]³⁺ by [Cr(CN)₆]³⁻, [Fe(CN)₆]³⁻ and [Ni(CN)₄]²⁻ in aqueous solutions as a function of alkali metal ions which were added for adjustment of ionic strength. The quenching rate constants in [Ru(NN)₃]²⁺-[Cr(CN)₆]³⁻ and [Cr(bpy)₃]³⁺-[Cr(CN)₆]³⁻ systems are changed by the cations as Li⁺>Na⁺>K⁺≈Rb⁺≈Cs⁺. On the other hand, the rate constants in [Ru(NN)₃]²⁺-[Fe(CN)₆]³⁻ and [Ru(NN)₃]²⁺-[Ni(CN)₄]²⁻ systems, which are diffusion-controlled reactions, are not varied by the alkali metal cations. The obtained order (Li⁺>Na⁺>K⁺≈Rb⁺≈Cs⁺) of the quenching rate constant is quite different from salt effects, Li⁺<Na⁺<K⁺<Rb⁺<Cs⁺, which have been obtained in the electron transfer reactions between complex anions.     

An improved synthesis, crystal structures, and metallochromism of salts of [Ru(tolyl-terpy)(CN)3]

The previously reported complex [Ru(ttpy)(CN)₃]⁻ [ttpy = 4′(p-tolyl)-2,2′:6′,2″-terpyridine] is conveniently synthesised by reaction of ttpy with Ru(dmso)₄Cl₂ to give [Ru(ttpy)(dmso)Cl₂], which reacts in turn with KCN in aqueous ethanol to afford [Ru(ttpy)(CN)₃]⁻ which was isolated and crystallographically characterised as both its (PPN)⁺ and K⁺ salts. The K⁺ salt contains clusters containing three complex anions and three K⁺ cations connected by end-on and side-on cyanide ligation to the K⁺ ions. The solution speciation behaviour of [Ru(ttpy)(CN)₃]⁻ was investigated with both Zn²⁺ and K⁺ salts in MeCN, a solvent sufficiently non-competitive to allow the added metal cations to associate with the complex anion via the externally-directed cyanide lone pairs. UV-Vis spectroscopic titration of (PPN)[Ru(ttpy)(CN)₃] with Zn(ClO₄)₂ showed a blue shift of 2900 cm⁻¹ in the ¹MLCT absorption manifold due to the ‘metallochromism’ effect; a series of distinct binding events could be discerned corresponding to formation of 4:1, 1:1 and then 1:3 anion:cation adducts, all with high formation constants, as the titration proceeded. In contrast titration of (PPN)[Ru(ttpy)(CN)₃] with the more weakly Lewis-acidic KPF₆ resulted in a much smaller blue-shift of the ¹MLCT absorptions, and the titration data corresponded to formation of 1:1 and then 2:1 cation:anion adducts with weaker stepwise association constants of the order of 10⁴ and then 10³ M⁻¹. Although association of [Ru(ttpy)(CN)₃]⁻ resulted in a blue-shift of the ¹MLCT absorptions, the luminescence was steadily quenched, as raising the ³MLCT level makes radiationless decay via a low-lying ³MC state possible.     

Requirement of different ionic concentrations for optimal translation of α and β globin mRNA in Krebs II ascites cell-free system

The translation of rabbit α globin mRNA in a Krebs II ascites cellfree system was more dependent upon the K⁺ concentration than rabbit β globin mRNA. The optimal KCl concentration was approximately 70 mM for the synthesis of the α chain and between 80 and 90 mM for that of the β chain. With CH₃ CO₂K the optimum concentration for α chain synthesis was also 70 mM but the optimum for the β chain synthesis was not sharp any more and ranged from 70 mM to over 110 mM. In the range of the optimal Mg²⁺ concentration for the α and β globin chain synthesis the $\text{α}\text{β}$ ratio decreased when the Mg²⁺ concentration increased. In the presence of DTT and EDTA the optimal KCl concentration for both α and β globin chain synthesis decreased.     

Determination of formation constants for complexes of very high stability: log β4 for the [Pd(CN)4] ion

The formation constant, log β₄ = 62.3 for [Pd(CN)₄]²⁻ is reported at 25 °C in 0.1 M NaClO₄. This value of log β₄ was determined using a competition reaction, monitored using UV-Vis spectroscopy and ¹H NMR. The competition reaction used was with the tetraamine ligand 2,3,2-tet(1,4,8,11-tetraazaundecane), for which log K₁ = 47.8 at 25 °C in 0.1 M NaClO₄ was determined by competition with thiocyanate, as described by earlier workers (Q.Y. Yan, G. Anderegg, Inorg. Chim. Acta 105 (1985) 121.). Also reported is a value of log β₄ for the [Pd(SCN)₄]²⁻ ion of 27.2 in 0.1 M NaClO₄, determined by competition with 2,2,2-tet. Measurement of log K₁ for cyclam with Pd(II) was attempted using a competition reaction with cyanide, combined with the very high value of log β₄ for [Pd(CN)₄]²⁻ measured here. It appeared that the equilibrium being followed was actually [Pd(cyclam)]²⁺ + 2CN⁻ ? [Pd(cyclam)(CN)₂], for which a constant of log K = 5.2 was obtained. ¹H NMR and IR studies suggested that the complex [Pd(cyclam)(CN)₂] was prone to oxidation to Pd(IV), followed by disproportionation to [Pd(cyclam)]²⁺ and, presumably, (CN)₂. The very high value of log β₄ for [Pd(CN)₄]²⁻ found here appears to be the highest formation constant known for any metal ion.     

The soluble [NiFe]-hydrogenase from<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> contains four cyanides in its active site,one of which is responsible for the insensitivity towards oxygen

Infrared spectra of ¹⁵N-enriched preparations of the soluble cytoplasmic NAD⁺-reducing [NiFe]-hydrogenase from Ralstonia eutropha are presented. These spectra, together with chemical analyses, show that the Ni-Fe active site contains four cyanide groups and one carbon monoxide molecule. It is proposed that the active site is a (RS)₂(CN)Ni(-RS)₂Fe(CN)₃(CO) centre (R=Cys) and that H₂ activation solely takes place on nickel. One of the two FMN groups (FMN-a) in the enzyme can be reversibly released upon reduction of the enzyme. It is now reported that at longer times also one of the cyanide groups, the one proposed to be bound to the nickel atom, could be removed from the enzyme. This process was irreversible and induced the inhibition of the enzyme activity by oxygen; the enzyme remained insensitive to carbon monoxide. The Ni-Fe active site was EPR undetectable under all conditions tested. It is concluded that the Ni-bound cyanide group is responsible for the oxygen insensitivity of the enzyme.Abbreviations BV benzyl viologen - DCIP 2,6-dichlorophenol-indophenol - EXAFS extended X-ray absorption fine structure - FTIR Fourier transform infrared - MV methyl viologen - SH soluble NAD⁺-reducing hydrogenase - XAS X-ray absorption spectroscopy     

Fluorescence spectra,fluorescence quantum yield and dissociation constant of sarafloxacin

In this study, the fluorescence spectra of sarafloxacin (SAR) under different pH conditions were investigated to determine the structural changes due to protonation that result from change in pH. At pH < 1.02, SAR exists in the H₃L²⁺ form for which the maximum fluorescence emission wavelength was about 455 nm. At pH 1.87–4.94, SAR exists in the H₂L⁺ form in which H₃L²⁺ loses one proton in the nitrogen molecule at the 1‐position in the quinoline ring. Fluorescence intensity was strong and steady and the maximum emission wavelength was 458 nm. At pH 7.14–9.30, the maximum emission wavelengths were gradually blue shifted to 430 nm with increase in pH, here SAR exists in the form of a bipolar ion HL in which H₂L⁺ loses a carboxyl group proton. At pH > 11.6, HL transforms into anionic L^? in which HL loses one proton from the piperazine ring, leading to a decrease in fluorescence intensity, and the maximum emission wavelength was red shifted to approximately 466 nm. The two‐step dissociation constant pKa for SAR was calculated, pK _a1 was 6.06 ± 0.37 and pK _a2 for SAR was 10.53 ± 0.19. In a pH 3.62 buffer solution with quinine sulfate as the reference, the fluorescence quantum yield of SAR at the maximum excitation wavelength of 276 nm was 0.09.