Mol Of Heme Research Articles

Oxygen Equilibrium of Hemoglobins Containing Unnatural Hemes: EFFECT OF MODIFICATION OF HEME CARBOXYL GROUPS AND SIDE CHAINS AT POSITIONS 2 AND 4

Stable ferro-forms of hemoglobins containing various hemes such as dimethyl proto-, dimethyl meso-, etio-, meso-, hemato-, and deuteroheme were prepared by combination of human globin with hemes, followed by reduction with dithionite and purification with carboxymethyl Sephadex column. Sedimentation coefficients of these hemoglobins were all near to 4.0 S and chemical analysis showed that 4 moles of heme were bound per mole of hemoglobin. Esterification of propionyl carboxyl groups of heme did not change the absorption spectra of hemoglobin; absorption spectra of dimethyl protohemoglobin were essentially identical with those of protohemoglobin, and absorption spectra of dimethyl mesohemoglobin with those of mesohemoglobin. Absorption spectra of etiohemoglobin, in which the propionic acid groups of mesoheme were substituted by ethyl groups, were similar to those of mesohemoglobin. Parameters of oxygen equilibrium, namely oxygen affinity, n in Hill's equation, and Bohr effect, of hemoglobin containing hemes which lack free carboxyl groups were the same as those for the corresponding hemoglobin with free propionic acid groups. Oxygen affinities of meso-, deutero-, hemato-, and protohemoglobin, in which heme side chains at positions 2 and 4 are ethyl, hydrogen, hydroxyethyl, and vinyl groups, respectively, were in the ratio of 5:2:1.3:1, corresponding to the reverse order of the negative inductive effects of the groups at positions 2 and 4. These results suggest the effect of π electron of a porphyrin ring on the oxygen binding.

The anaerobic interaction of ferrocytochrome c with the “ferric” and “oxygenated” forms of purified cytochrome c oxidase. XVI. Cytochrome oxidase and its derivatives

1. The anaerobic interaction of ferrocytochrome c with “ferric” cytochrome c oxidase (EC 1.9.3.1) in the absence of ascorbate or ferro/ferricyanide cannot be interpreted as the reaction of a uniform ferric oxidase. The increase of the α-band at 605 mμ precedes that of the γ-band at 444 mμ and the γ/α ratio increases over a period of several minutes. 2. The biphasic form of the reaction is far less noticeable in the reaction of “oxygenated” oxidase with ferrocytochrome c. A similar difference has been previously found in the reduction by dithionite. 3. A spectrophotometric method is developed which allows calculations of the concentrations of ferrous and ferric cytochromes a, a 3 and c at various times after mixing oxidase and ferrocytochrome c. This is based on the extinction coefficients of cytochrome c and those of ferro- and ferricytochromes a and a 3 (refs. 1 and 2) at 605, 550 and 444 mμ. 4. With ferric oxidase, rapid reduction of ferricytochrome a is followed by a far slower reduction of ferricytochrome a 3 by ferrocytochromes c and a. No rapid equilibrium is established. Two equivalents of ferrocytochrome c are required for the reduction of one ferricytochrome a and two for that of ferricytochrome a 3 (4 per 2 moles haem a). The additional electron acceptor is probably the intrinsic copper of the oxidase. 5. With oxygenated oxidase, initial rapid conversion of the ferryl form of cytochrome a 3 is suggested by the fact that initially more than two equivalents of ferrocytochrome c are oxidized per mole of haem a. The ferricytochrome thus formed ( a 3 x 3+, ref. 2) is spectroscopically similar to or identical with ferricytochrome a 3 3+, but differs from it by its far more rapid reduction by ferrocytochromes c and a. 6. Ferricytochrome a 3 x 3+ probably plays a larger role in the enzymic reaction of the oxidase than does the ferricytochrome known as a 3 3+ present in the ferric oxidase preparation.

Cytochrome oxidase and its derivatives. VII. Further observations on the compound formed by the reaction of ferrous cytochrome c oxidase with oxygen and hydrogen peroxide and on its reactions

Molecular O 2 and H 2O 2 acting upon ferrous cytochrome c oxidase (EC 1.9.3.1) appear to form the same compound. Under conditions of complete formation by O 2 or by H 2O 2, the Soret band lies at 427–428 mμ and is of about equal height to the Soret band of the ferric oxidase at 418 mμ. This makes the interpretation of its absorption curve as one of a mixture of ferrous and ferric oxidase untenable. While formation of the 428-mμ compound is complete when 3 moles of H 2O 2 are added to 1 haem a equivalent of oxidase reduced by 4 moles of dithionite a greater excess of molecular O 2 (8–20 moles per mole of haem a) must be added to the gas phase. The 428-mμ compound can, however, be formed under conditions under which no H 2O 2 is formed by the autoxidation of the reductant, e.g. formamidinosulphinic acid or 1 mole of ferrocytochrome c. The effects of various conditions (standing in air and after evacuation) and reagents (CO, ferricyanide and catalase) are studied. They show that the compound cannot be a reversibly oxygenated compound such as oxyhaemoglobin.

On the mechanism of sulfide inactivation by methemoglobin

Methemoglobinemia induced in laboratory mammals protects them against death from inorganic sulfide poisoning. Similarly, an intraperitoneal depot of human methemoglobin prepared in vitro protects mice against a subsequent intraperitoneal injection of sodium sulfide. Human methemoglobin is effective whether in simple solution or in red cells; oxyhemoglobin is inactive under similar circumstances. The prophylactic efficacy of methemoglobin is presumably due to its ability to bind or otherwise inactivate sulfide. From the displacement of the dose-mortality curve in methemoglobinemic mice it is inferred that 2–4 moles of sulfide are inactivated in vivo for each mole of methemoglobin heme. The comparable ratio in poisoning by cyanide is about one, and for high doses of azide the ratio also approaches unity. That the ferric iron of methemoglobin binds (and perhaps otherwise inactivates) sulfide is deduced from the observation that, when methemoglobin is converted to cyanmethemoglobin, its antidotal effectiveness is reduced by the equivalent of at least 1 mole of sulfide per mole of heme. The inference that methemoglobin possesses more binding sites for sulfide than for cyanide is proved by the demonstration that cyanmethemoglobin protects mice against sulfide poisoning. These extra binding sites must be associated with some part of the molecule other than the heme iron, which is known to be occupied by cyanide. To be effective against sulfide poisoning, however, cyanmethemoglobin must be prepared from nitrite-treated hemoglobin, not from ferricyanide-treated hemoglobin. Therefore, the former but not the latter must possess sulfide-binding sites unavailable to cyanide. On the assumption that spectral changes reflect only 1:1 interactions between anion and heme iron, dissociation constants have been calculated from spectrophotometric titration curves. They indicate that the hydrosulfide ion (HS −) is bound to the ferric iron more tightly in nitrite-treated hemoglobin than in ferricyanide-treated hemoglobin. In contrast, no difference between these two pigments could be demonstrated in their affinities for cyanide or azide. Another difference between nitrite-treated and ferricyanide-treated hemoglobins is the presence of at least one more free sulfhydryl group in the nitrite-prepared pigment.

On cytochrome c oxidase. I. The extinction coefficients of cytochrome a and cytochrome a3.

Summary 1. In agreement with Gibson, a shoulder at 420–424 m μ is present in the absorption spectra of preparations of cytochrome c oxidase (ferrocytochromec: oxygen oxidoreductase, EC 1.9.3.1) reduced with Na 2 S 2 O 4 . An examination of the spectra in the presence and absence of cyanide showed that this is due to the presence of equal amounts of non-reducible cytochrome a and non-reducible cytochrome a 3 . 2. Cytochrome c oxidase was titrated with NADH and phenazine metho-sulphate. It was found that each mole of NADH reduced 1 mole of haem a and 1 atom of copper. 3. Functionally inactive copper present in the enzyme preparation, added Cu(II) and Fe(III) salts and mitochrome, a modification product of cytochrome c oxidase, are not reduced by phenazine methosulphate, under the conditions of the titration. 4. The values of Δem M (reduced minus oxidized) at 605 m μ , and 445 m μ , respectively, were: cytochrome a , 19.4 and 82; cytochrome a 3 , 4.6 and 82; cytochrome aa 3 , 24.0 and 164. 5. Concentrations of cytochrome a, calculated on the basis of the widely used value of 16.5 for Δem M ( A 605 m μ A 630 m μ ), are 150% too high.

Cytochrome oxidase and its derivatives. VI. The CO combining capacity of cytochrome C oxidase.

Summary Ferrous cytochrome a 3 is known to bind CO at pH 7.4, whereas cytochrome a does not do so. It is found that about one-half of the total haem a of cytochrome c oxidase (EC 1.9.3.1) is able to bind CO. This shows that the ratio of cytochrome a to cytochrome a 3 is about 1. The validity of the method was established by showing that haemoglobin bound 1 mole of CO per mole of protohaem. When cytochrome oxidase is kept at pH 10 at room temperature both cytochromes a and a 3 are modified and both the modified cytochromes now react with CO. It is found that 1 mole of haem a of the modified oxidase binds approx. 1 mole of CO. This lends additional support to the result of the experiments with native cytochrome oxidase.