Horse Metmyoglobin Research Articles

The Kinetics and Mechanism of Reduction of Electron Transfer Proteins and Other Compounds of Biological Interest by Dithionite

Abstract The kinetics of the reduction by dithionite of several biochemically interesting substances has been investigated under anaerobic conditions by stopped flow spectrophotometry at pH 8.0 and 25°. Solutions of sodium dithionite were prepared and standardized by titration against lumiflavin 3-acetate in the absence of oxygen. Under pseudo-first order conditions (dithionite in excess) the reactions of spinach ferredoxin, horse metmyoglobin, lumiflavin 3-acetate, horse heart ferricytochrome c, and spinach plastocyanin conform to first order kinetics for several half-lives. Dependence of the observed pseudo-first order rate constant (kobs) on the dithionite concentration is described by the equation, kobs = a[S2O42-]½, where a = 8.6, 100, and 960 m-½ s-1 for ferredoxin, metmyoglobin, and lumiflavin 3-acetate, respectively. These results support a mechanism involving SO2- as the kinetically important reducing species. With cytochome c and plastocyanin, the dependence of kobs on dithionite concentration is described by the equation, kobs = a[S2O42-]½ + b[S2O42-], where a and b are 1450 m-½ s-1 and 1.5 x 104 m-1 s-1 in the case of cytochrome c and 1100 m-½ s-1 and 1.35 x 105 m-1 s-1 in the case of plastocyanin. These two proteins apparently react with both SO2- and S2O42-. Analysis of the reaction of cytochrome c with dithionite in the presence of oxygen gave an estimated rate constant of 47 m-½ s-1 for the oxygen-dithionite reaction. The rate constant of the reaction of ferricyanide with S2O42- is 1.4 x 105 m-1 s-1. Evidence is presented that ferricyanide also reacts rapidly with SO2-. When dithionite reacts with excess ferricytochome c, lumiflavin 3-acetate, or oxygen, the reaction rate is virtually independent of the oxidant concentration and a limiting first order rate constant of 1.7 s-1 is approached in each case. This rate constant has been assigned to the monomerization rate of S2O42-, and its temperature dependence gives an activation energy of 24.1 Cal per mole for the dissociation of S2O42-. Determination of the equilibrium constant (K) for the reaction S2O42- ⇌ 2SO2- by electron paramagnetic resonance spectroscopy has given a value of 1.4 ± 0.4 x 10-9 m. Dividing the above listed values of a by K½ gives the absolute rate constants for the reactions between SO2- and oxidants.

Amino acid free radicals in oxidised metmyoglobin

1. 1.|A technique is described for distinguishing by electron spin resonance (ESR) spectrometry between aromatic and aliphatic amino acids. It is employed to determine the nature of the free radicals which are formed when metmyoglobin or methaemoglobin is oxidized by H 2O 2. The results obtained strongly suggest that the first free radical produced is that of a phenylalanine or histidine residue close to the porphyrin on the distal side, and that the free radical site is rapidly transferred to a tyrosine residue. 2. 2.|Ultraviolet absorption spectrophotometry shows that in the presence of excess H 2O 2 some of the aromatic units are converted to DOPA and DOPAquinonelike residues, but the two tryptophan units are not quick to oxidize. 3. 3.|Horse methaemoglobin behaves qualitatively like horse metmyoglobin during oxidation but the free radicals are destroyed more rapidly. Whale metmyoglobin differs significantly from horse metmyoglobin; the differences are probably explainable in terms of the protruding tyrosine residue in the former. 4. 4.|The preservation of myoglobin against oxidation damage in vivo is attributed to the existence of a means of transferring free radical sites from the vicinity of the haem to a tyrosine residue which is accessible to external reducing agents. 5. 5.|Under the specific conditions used for generation of free radical sites in individual amino acids and in peptides the greatest ease of formation and stability are observed in the radicals from tyrosine, tryptophan and the products of their hydroxylation. ESR signals due to hydroxy-aromatic radicals display a strong tendency to saturate which is increased by further hydroxylation. As first formed the phenylalanine radical does not give a saturated signal, but in the presence of excess H 2O 2 it undergoes further oxidation and is therefore recognized as aromatic by the saturated signal of the final product.

A comparison of the conformation of sperm whale metmyoglobin with that of apomyoglobin

Sedimentation and diffusion coefficients have been determined for horse and sperm whale metmyoglobins and apomyoglobins; the values obtained for horse metmyoglobin and apomyoglobin are closely similar to those determined for sperm whale metmyoglobin and apomyoglobin, respectively. The intrinsic viscosities of sperm whale metmyoglobin and apomyoglobin have also been estimated. A comparison of the physical properties of sperm whale metmyoglobin with those of apomyoglobin showed that removal of the haem group resulted in a decrease in the sedimentation and diffusion coefficients and an increase in the intrinsic viscosity. Molecular weights and frictional ratios of sperm whale metmyoglobin and apomyoglobin were calculated from the results; the molecular weights were in good agreement with the known empirical molecular weights of the proteins. The frictional ratio of apomyoglobin was significantly greater than that of metmyoglobin. It is concluded that the shape of apomyoglobin is either more swollen or more asymmetrical than that of metmyoglobin.

Investigations on the influence of hemin and hemin derivatives on the nuclear-magnetic relaxation of water protons

Haemin, haemin derivatives and ferric haemoproteins are investigated on their influence on the nuclear-magnetic relaxation of water protons. The effectiveness increases in the following order: ferricytochrome c (neutral form), horse methaemoglobin, horse metmyoglobin, cheironomus methaemoglobin, ferricytochrome c (acidic form), haematin. The high effectiveness of the haematin is already decreased by the adsorptive bonding to polyvinylpyrrolidone. The influence on the relaxation of water protons may be divided into a magnetic and a steric component, the latter is thought to be a relative measure for the accessibility of the prosthetic groups in the haemin derivatives. It is shown, that the haemin groups in cytochrome c and cheironomus methaemoglobin are situated at the surface more than those of horse methaemoglobin or horse metmyoglobin. By the bonding of ligands to the horse methaemoglobin the influence of the iron on the relaxation of water protons is attributable not only to the variation of the magnetic moment but also to the variation of the steric arrangement of the prosthetic group to the protein. The methaemoglobin fluoride compound shows especially striking behaviour.

The crystal structure of horse met-myoglobin - I. General features: the arrangement of the polypeptide chains

The preliminary results of an X-ray study of crystalline horse met-myoglobin are described. This protein crystallizes in the monoclinic space-group P2 1 with two molecules in general positions in the unit cell. The optical properties of the crystals show that the haem groups are parallel and lie approximately in the plane containing b and the internal bisector of β. a and c Patterson projections contain ridges of high vector density perpendicular to b and 15*4A apart; it is deduced that in real space the contents of the cell are arranged in pronounced layers of alternately high and low electron density, also perpendicular to b and 15*4 A apart. The b projections also exhibit rods of high vector density, 9*5 A apart and inclined at 20° to a . These rods are identified as the vector equivalents of the polypeptide chains, which thus run parallel to [20T] and 9*5 A apart. A Patterson projection along [20T] shows the rods well-resolved and in end-on view; by making certain plausible assumptions a Fourier projection has been made along the same direction. During shrinkage the cell dimensions change in such a way that the inter-chain distance alters very little; it is deduced that the molecules have flat sides parallel to (102) and that the liquid of crystallization is situated mainly on their [20T] faces. During shrinkage liquid is withdrawn and the molecules slide past one another along their flat sides. It is not possible to determine the whole shape of the molecule unequivocally; the most plausible model is a platelet built of four parallel co-planar lengths of chain each 54 A long (see figure 13). There are striking analogies between the structures of myoglobin and of horse haemoglobin. The type of chain folding seems to be the same in the two proteins, and probably the same as in a-keratin; and it may be that myoglobin has a structure analogous to that of one of the four layers of which haemoglobin is believed to be built up, the molecular weights of the two proteins being in the ratio 1:4.