Myoglobins oxidation-reduction

The abihty of iron to exist in two stable oxidation states, ie, the ferrous, Fe ", and ferric, Fe ", states in aqueous solutions, is important to the role of iron as a biocatalyst (79) (see Iron compounds). Although the cytochromes of the electron-transport chain contain porphyrins like hemoglobin and myoglobin, the iron ions therein are involved in oxidation—reduction reactions (78). Catalase is a tetramer containing four atoms of iron peroxidase is a monomer having one atom of iron. The iron in these enzymes also undergoes oxidation and reduction (80). 

The first electrochemical studies of Mb were reported for the horse heart protein in 1942 (94) and subsequently for sperm whale Mb (e.g., 95) through use of potentiometric titrations employing a mediator to achieve efficient equilibriation of the protein with the electrode (96). More recently, spectroelectrochemical measurements have also been employed (97, 98). The alternative methods of direct electrochemistry (99-102) that are used widely for other heme proteins (e.g., cytochrome c, cytochrome bs) have not been as readily applied to the study of myoglobin because coupling the oxidation-reduction eqiulibrium of this protein to a modified working electrode surface has been more difficult to achieve. As a result, most published electrochemical studies of wild-type and variant myoglobins have involved measurements at eqiulibrium rather than dynamic techniques. 

Recent work has resolved some of the issues that complicate direct electrochemistry of myoglobin, and, in fact, it has been demonstrated that Mb can interact effectively with a suitable electrode surface (103-113). This achievement has permitted the investigation of more complex aspects of Mb oxidation-reduction behavior (e.g., 106). In general, it appears that the primary difficulty in performing direct electrochemistry of myoglobin results from the change in coordination number that accompanies conversion of metMb (six-coordinate) to reduced (deoxy) Mb (five-coordinate) and the concomitant dissociation of the water molecule (or hydroxide at alkaline pH) that provides the distal ligand to the heme iron of metMb. 

The kinetics of myoglobin oxidation and reduction have been studied by a variety of experimental techniques that include stopped-flow kinetics, pulse radiolysis, and flash photolysis. In considering this work, attention is directed first at studies of the wild-type protein and then at experiments involving variants of Mb. 

Both siroheme enzymes form ferroheme-NO complexes in which the g value anisotropy appears somewhat smaller than in the corresponding complexes of most other enzymes. The EPR spectra of the complexes somewhat resemble the spectra of the high-temperature myoglobin-NO complexes. The hyperfine splitting from the NO nitrogen nucleus is evident at intermediate g values but is not well resolved. These enzymes are capable of reducing NO to ammonia if supplied with low potential reducing equivalents. Other heme proteins also catalyze oxidation reduction reactions with NO. 

Direct recording of the respiratory pigments in situ, by absorbance and/or fluorescence spectroscopy, provides one way of resolving this dilemma. In anaesthetized animals spectroscopy of exposed tissues 24, 25) reports the oxidation-reduction state of the electron transport chain and the oxygenation of haemoglobin/myoglobin. Unfortunately the rapid fluctuations of O2 delivery and consumption make it difficult to establish convenient steady states. An alternative procedure is to freeze the tissue rapidly and then to obtain spectra more leisurely at low temperatures. 

The function of iron in the body depends on the compound in which iron occurs. Iron mostly participates in oxygen transport through the bloodstream and oxygen storage in muscle tissue (the iron is in the haemoglobin and myoglobin) and participates in catalytic and oxidation-reduction reactions (iron in haem and flavin enzymes). 

Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Fig. 8.9 UV—Vis spectra of intercalated biomolecules assembled AMP. Soret band absorptions for (A) oxidized myoglobin (met-Mb) and after dithionite reduction (deoxy-Mb), and (B) after CO (CO-Mb) and 02 bindingto intercalated deoxy-Mb.

J.F. Stargardt, F.M. Fiawkridge, and H.L. Landrum, Reversible heterogeneous reduction and oxidation of sperm whale myoglobin at a surface modified gold minigrid electrode. Anal. Chem. 50, 930-932 (1978). 

Mb. Subsequent application of this technique to reduction of various derivatives of reduced and oxidized myoglobin led to the observation that the rate of reduction by hydrated electrons depends primarily on the net charge of the protein and the dissociation constant for formation of ligand bound derivatives of metMb. 

Fox, J. B., and Ackerman, S. A. (1968). Formation of nitric oxide myoglobin Mechanisms of the reaction with various reductants. J. Food Set. 33, 364-370. 

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