Cytochrome reactions, horse-heart

Cytochrome c is a single-heme protein, MW = 12 kDa, fairly universal in respiratory ET chains [77]. The reduction potential is 265 mV (SHE, horse heart). ET reactions in homogeneous solution and promoter-induced reversible cyclic voltammetry are well characterized. Horse heart cyt c has been in recent focus in some of the first STM investigations of metalloprot-... 

Table II. Slope of Brpnsted—Bjerrum Plot for Some Horse-Heart Cytochrome c Reactions (25 °C)...

Prince, R. C., Cogdell, R. J., and Crofts, A. R., 1974, The photo-oxidation of horse heart cytochrome c and native cytochrome 2 by reaction centers from Rhodopseudomonas spheroides R26. Biochim. Biophys. Acta, 347 lnl3. 

The ability of clathrochelates to form ion pairs and covalently attached complexes is utilized in biochemistry [315-321], The stereoselectivity of the redox reactions of plastocyanine and horse heart cytochrome C with several cage complexes was reported in Ref 319. Studies on stereoselective electron transfer in different systems provide information on the importance of close ion pair association of a cage complex with protein in chiral discrimination. 

Direct electrochemistry has also been used (72-78) to couple the electrode reactions to enzymes for which the redox proteins act as cofactors. In the studies, the chemically reduced or oxidized enzyme was turned over through the use of a protein and its electrode reaction as the source or sink of electrons. In the first report (72, 73) of such application, the electrochemical reduction of horse heart cjd,ochrome c was coupled to the reduction of dioxygen in the presence of Pseudomonas aeruginosa nitrite reductase/cytochrome oxidase via the redox proteins, azurin and cytochrome C551. The system corresponded to an oxygen electrode in which the four-electron reduction of dioxygen was achieved relatively fast at pH 7. 

RC Prince, RJ Cogdell and AR Crofts (1974) The photooxidation of horse heart cytochrome c and native cytochrome Cj by reaction centres from Rhodopseudomonas sphaeroides R-26. Biochim Biophys Acta 347 1-13... 

In contrast to other systems in which the mechanism (inner-sphere or outer-sphere electron transfer) may be a matter of debate, the reaction between the protein horse heart cytochrome c with anionic Cu" complexes was adjudged to proceed by an outer-sphere mechanism. [170] The copper(ll) complex bis(5,6-bis(4-suphonatophenyl)-3-(2-pyridyl)-l,2,4-triazine)Cu(ll), (the ligand is commonly known as ferrozine) possesses square pyramidal geometry with the two bidentate ligands in the equatorial plane, and the fifth axial position is occupied by a water molecule, in aqueous solution. 

In recent publications (5, 6), we described studies of the electron transfer reaction between horse heart cytochrome c and synthetic rat liver cytochrome fcs. We observed a rate constant for electron transfer of 4 X 10 s" in low ionic strength solutions in which these proteins form a strong 1 1 complex... 

Fig. 4. Imidazole concentration dependence of the rate of binding of imidazole to ferricytochrome c. A solution of 7.5 /aM horse heart cytochrome c was mixed with solutions containing known concentrations of imidazole in a stopped-flow apparatus. The buffer was 0.1 M sodium borate pH 9.0. The reaction was followed at 410 nm, temperature 21 C. The inset depicts the mechanism by which the sulphur of methionine (80) dissociates from the central iron and is subsequently replaced by a nitrogen atom of imidazole.

Optimal activity of the purified enzyme solubilized in Triton X-100 is obtained in the presence of excess phospholipids. The pH optimum of the steady-state reaction with horse heart ferrocytochrome c occurs at pH 6, yielding a turnover of about 80 electrons/sec, similar to the value obtained for the enzyme from P. denitrificans. Remarkably, a purified membrane-bound c cytochrome, identified by its N-terminal sequence as cytochrome Ci from the 6c 1 complex, stimulates the rate of electron transfer between horse heart c5d ochrome c and the cytochrome c oxidase by about a factor of two. The in vitro enzyme assay with purified cytochrome oxidase and reduced amicyanin showed no activity only after the addition of endogenous cytochrome C550 (or horse heart cjfto-chrome c) did oxidation of amicyanin occur, in agreement with the sequence of electron transfer ami — cjft C550 CCO. 

Ill (140). The ET reaction is initiated by photogenerated [Ru(bpy)3], which rapidly reduces the surface ruthenium. The [Ru(bpy)3] + is then scavenged by EDTA before it can back react with a5Ru(II)(histidine). Electron transfer to the protein metal center is then monitored spectroscopically. In the case of a heme (FeP), a fast increase in absorbance because of direct reduction of Fe(III)P by [Ru(bpy)3] is followed by a slower increase in absorbance due to reduction of Fe(III)P by the Ru(II) on the protein surface. Control flash experiments with unmodified proteins show only the fast initial increase in absorbance resulting from Fe(III)P reduction by [Ru(bpy)3]-. Such control experiments demonstrate for horse heart cytochrome c (140), azurin (93), and sperm whale myoglobin (30) that slow reduction of the heme by the EDTA radical produced in the scavenging step does not occur in competition with intramolecular ET. For C. krusei cytochrome c, however, the control experiment shows evidence for slow EDTA radical reduction of the heme after initial fast reduction by [Ru(bpy)3p (164). 

Conformational changes could control ET reactions in proteins. The rates of such changes often are in the same range as ET rates for example, the T-R transition in hemoglobin occurs at a rate of approximately 2 X 10 s (112). Hoffman and Ratner (66,67) have pointed out that a way to test for conformational control of an ET reaction is to measure the reaction rate at different driving forces. If the rate stays the same, the ET reaction is conformationally controlled. If it does not, it is not confor-mationally controlled. No evidence for conformation control exists for ET in ruthenium-modified proteins on this basis. Data from both ruthenated His-33 in horse heart cytochrome c (126) and ruthenated His-48 in myoglobin (103) show that the rate changes with AG° in a manner consistent with Marcus theory. 

The carboxymethylation of horse heart cytochrome c by [2-i C] bromo-acetate has been monitored by NMR spectroscopy (77). In the absence of cyanide ion position 65 of the cytochrome c is the only reactive methionine. Carboxymethylation gives as expected a single resonance at 48.4 ppm (similar to carboxymethylated methionine peptides). In the presence of cyanide ion the methionines at positions 65 and 80 are both reactive and two resonances for the carboxymethylated product were observed 48.4 ppm position 65 48.2 ppm position 80. Upon reduction of the iron and carboxymethylation, the position 80 carboxymethylmethionine moves to 48.4 ppm, suggesting that the carboxymethylmethionine-80 feels the oxidation state of the metal (Fe +-Fe +). At long reaction times further carboxymethylations were detected. 

Assay Methods. 2-Oxoacid ferredoxin oxidoreductase activity is determined by following the absorbance at 550 nm, due to the ferredoxin-dependent reduction of horse heart cytochrome c (Sigma Chemicals, St. Louis, MO) in the presence of 2-oxoacid substrates, essentially as described by Kerscher et al. The assay is conducted at 50°, in 10 mAf potassium phosphate buffer, pH 6.8, in the presence of 2-4 mAf 2-oxoacids (2-oxoglutarate purchased from Nacalai Tesque, Japan, was mainly used), 50-100 p,Af coenzyme A (Kohjin, Japan), 17 pg of the Sulfolobus zinc-containing ferredoxin (purified as described above), 50 pAf horse heart cytochrome c (Sigma Chemicals), and an appropriate amount of enzyme, in a total volume of 1 ml. The reaction is initiated by addition of the enzyme, and nonenzymatic reduction of cytochrome c by coenzyme A at this temperature is... 

Poly-L-lysine inhibits the electrode reaction of native horse heart cytochrome c, as shown by dc voltammetry (Figure 3), again analogous to its inhibiting effect on the cytochrome c-oxidase reaction. The effect on the ac cyclic voltammetry peak current, //,(ac), is more marked. The varation with poly-L-lysine concentration is consistent with adsorption of poly-L-lysine onto the electrode surface, decreasing the effective free electrode area. 

Cytochrome c in the solution often exhibits an irreversible voltammetric response at gold electrodes, with the exception of a carefully prepared gold electrode [32]. A strong adsorption of cyt. c on an electrode surface is considered to block the ET reaction of cyt. c in the solution. Hin-nen and coworkers, and Hinnen and Nild measured the formal potential of horse heart cyt. c adsorbed on gold, ruthenium, and glassy carbon electrodes by... 

Thin-layer electrochemistry with an optically transparent electrode (OTE) enables simultaneous monitoring of both the electrochemical and optical responses of the system [1-3]. The oxidation state of the electroactive species in a cell can be precisely controlled by regulating the potential of the OTE and the species in the cell can be completely electrolysed within a short time (typically 20-120s). The electrochemical technique combined with a gold minigrid OTE was first applied to biological molecules to characterize the thermodynamic parameters of the redox reaction of horse heart cytochrome c in the presence of redox mediators [4]. 

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