Aggregation in five-coordinate high-spin natural hemins: determination of solution structure by proton NMR

1H NMR measurements (at 500 MHz) of nuclear spin-spin relaxation time T2 (from NMR line width) at different temperatures are reported for aggregates of several five-coordinate high-spin iron(III) complexes of proto-, deutero-, and coproporphyrins in solution and are utilized to determine their solution structure. Extensive aggregation of these complexes in solution is observed, and the dominant form of the aggregates is shown to be dimers. The degree of aggregation for these iron(III) porphyrins follows the order proto- » deutero- > copro-. The line width of the heme methyl resonances was analyzed by using a nonlinear least-squares fit program working in finite difference algorithm. The values of T2 were used to determine the structural details of the dimer

Stability and characterization of iron(III) and iron(II) heme peptides encapsulated in aqueous detergent micelles: proton NMR and UV-visible spectroscopic studies

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Six-coordinated high-spin models for ferric hemoproteins: NMR and ESR study of the diaquo(protoporphyrinato IX)iron(III) cation and aquohydroxo(protoporphyrinato IX)iron(III) intercalated in aqueous detergent micelles

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Binding of cyanide and thiocyanate to manganese reconstituted myoglobin and formation of peroxide compound: optical spectral, multinuclear NMR, and kinetic studies

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Heme CD as a probe for monitoring local structural changes in hemeproteins: alkaline transition in hemeproteins

Structural change due to acid-alkaline transition in hemeproteins were monitored by circular dichroism measurements in the Soret region. It was observed that in cytochrome c and horseradish peroxidase, alkaline transition results in a large change in the heme CD due to significant conformational change in the heme cavity region. In metmyoglobin a simple protolytic mechanism associated with alkaline transition involves very small conformational changes

Reaction of hydrogen peroxide and peroxidase activity in carboxymethylated cytochrome c: spectroscopic and kinetic studies

The peroxidase activity of carboxymethylated cytochrome c (Cmcytc) has been investigated by spectroscopic and kinetic techniques to examine the effect of carboxymethylation on the peroxidase activity of native cytochrome c (cytc). The optical spectrum suggests that the reaction of Cmcytc with H2O2 proceeds through only one intermediate, compound I. The apparent rate constant (kapp) for the reaction was found to be 17, 72 and 210 M−1 s−1 at pH 7.0, 5.0 and 3.5 respectively. These values are about 60 times larger than those reported for native cytc (0.236 M−1 s−1 at pH 7.0), and about five orders of magnitude lower than those for classical peroxidases. Cmcytc was found to catalyse oxidation of organic and inorganic substrates. The second order rate constant for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) by Cmcytc (205 [H2O2] s−1) is found to be larger than the corresponding value for native cytc (50 [H2O2] s−1) at pH 6.0. The carboxymethylation of cytc ruptures the Fe-S (Met 80) bond and increases the rate of its reaction with H2O2, and its catalytic activity. The specific activity of Cmcytc was measured spectrophotometrically by the reported method using ABTS as substrate, and was found to be 288, 473 and 872 μM min−1 mg−1 at pH 7.0, 5.0 and 3.5 respectively. Resonance Raman studies indicated the presence of a bis-histidine coordinated form of Cmcytc at neutral pH, and the existence of a population distribution of different ligation states such as bis-histidine (HH), histidine-water (HW) and five coordinate (5C) forms at lower pH. The relative population of different species in Cmcytc was found to be HH (~100%, ~50%, ~44%), HW (~0%, ~44%, 41%) and 5C (~0%, ~6%,15%) at pH 7.0, 4.7 and 3.1 respectively. We have attempted to correlate the pH dependence of the reaction of Cmcytc with hydrogen peroxide and its peroxidase activity with the haem stereochemical structures observed for Cmcytc. Steady-state and time-resolved tryptophan fluorescence studies on Cmcytc were done to probe the conformational changes around the haem pocket of Cmcytc

Follow-up of potential novel Graves' disease susceptibility loci, identified in the UK WTCCC genome-wide nonsynonymous SNP study

A recent association scan using a genome-wide set of nonsynonymous coding single-nucleotide polymorphisms (nsSNPs) conducted in four diseases including Graves' disease (GD), identified nine novel possible regions of association with GD. We used a case–control approach in an attempt to replicate association of these nine regions in an independent collection of 1578 British GD patients and 1946 matched Caucasian controls. Although none of these loci showed evidence of association with GD in the independent data set, when combined with the original Wellcome Trust Case–Control Consortium study group, minor differences in allele frequencies (P⩾10−3) remained in the combined collection of 5924 subjects for four of the nsSNPs, present within HDLBP, TEKT1, JSRP1 and UTX. An additional 29 Tag SNPs were screened within these four gene regions to determine if further associations could be detected. Similarly, minor differences only (P=0.042–0.002) were detected in two HDLBP and two TEKT1 Tag SNPs in the combined UK GD collection. In conclusion, it is unlikely that the SNPs selected in this replication study have a significant effect on the risk of GD in the United Kingdom. Our study confirms the need for large data sets and stringent analysis criteria when searching for susceptibility loci in common diseases