M.A. Proskurnin – Ph.D. (Chem.), Professor, Department of Analytical Chemistry, Faculty of Chemical,  Lomonosov Moscow State University

E-mail: proskurnin@gmail.com

M.E. Muratova – Student, Department of Chemistry, Lomonosov Moscow State University

E-mail: frau.muratova@gmail.com

A.V. Brusnichkin – Post-graduate Student, Faculty of Chemical, 

Lomonosov Moscow State University

E-mail: lenamy@list.ru

P.A. Gorkin – Post-graduate Student, Vernadsky Institute of Geochemistry 

and Analytical Chemistry of the RAS (Moscow) 

E-mail: pagorkin@mail.ru

D.S. Volkov – Ph.D. (Chem.), Junior Research Scientist, Department of Analytical Chemistry, 

Faculty of Chemistry, Lomonosov Moscow State University. Moscow

E-mail: dmsvolkov@gmail.com

E.V. Proskurnina – Ph.D. (Chem.), Associate Professor, Department of Medical Biophysics,  Faculty of Fundamental Medicine, Lomonosov Moscow State University; Senior Research Scientist,  Radiologic Diagnostics Department, Medical Research and Education Center  of the Lomonosov Moscow State University

E-mail: proskurnina@gmail.com

Yu.A. Vladimirov – Full Member of the RAS, Head of the Department of Medical Biophysics, 

Faculty of Fundamental Medicine, Lomonosov Moscow State University

E-mail: yuvlad@mail.ru

The conditions for the reduction of cytochrome c were selected. It was found that the reduction reaction with ascorbate proceeds slowly and a certain time is necessary for the reaction to proceed. It depends on the concentration of ascorbate. The optimum ratio of the concentrations of cytochrome c and ascorbate was selected. The optimum ratio was equal to 1: 5. The effect of sodium dodecyl sulfate (SDS) on cytochrome c associated with the oxidation of reduced cytochrome, was investigated. This effect is not described in the literature, and the ratio of concentrations of cytochrome and SDS is not justified. It has been shown experimentally that when the ratio of cytochrome c and SDS concentration is more than 1:80, the reduced cytochrome c is oxidized. This allows us to select the optimum ratio of the concentrations of cytochrome c and SDS, and a ratio of 1:50 was selected.

While bubbling CO into a solution of cytochrome c, the denaturation of the latter was observed, which is not mentioned in previous papers on cytochrome c-CO complex. It is concluded that it occurs at the interface. In a CO-saturated water (without gas bubbling), the complex is not formed. Because of this, when selecting the CO bubbling rate, it is necessary to take into account two opposite factors: the degree of formation of the complex, which increases with the rate and the denaturation of the protein, which also increases. 

Two-dimensional correlation spectroscopy (2DCOS) is used to assess whether the proposed conditions correspond to the production of the CO complex of ferrocytochrome c only and to what extent protein denaturation occurs in these conditions – a process that cannot be unambiguously estimated from the spectra. The difference between the conditions for obtaining the complex in the presence of SDS and tetraoleoylcardiolipin are shown. 

It was shown that the complex of cytochrome c with CO is unstable, the kinetics of its decomposition was measured, and the kinetic equation was obtained by approximating the obtained curve.

1. Petrosillo G., Ruggiero F.M., Paradies G. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria // The FASEB Journal. 2003. V. 17. № 15. P. 2202–2208.
2. Barr D.P., Mason R.P. Mechanism of radical production from the reaction of cytochrome c with organic hydroperoxides.: An esr spin trapping investigation // J. Biol. Chem. 1995. V. 270. № 21. P. 12709–12716.
3. Chung H.-T., Pae H.-O., Choi B.-M., Billiar T.R., Kim Y.-M. Nitric oxide as a bioregulator of apoptosis // Biochemical and Biophysical Research Communications. 2001. V. 282. № 5. P. 1075–1079.
4. Bilban M., Haschemi A., Wegiel B., Chin B.Y., Wagner O., Otterbein L.E. Heme oxygenase and carbon monoxide initiate homeostatic signaling // J. Mol. Med. 2008. V. 86. № 3. P. 267–279.
5. Boczkowski J., Poderoso J.J., Motterlini R. Co–metal interaction: Vital signaling from a lethal gas // Trends Biochem. Sci. V. 31. № 11. P. 614–621.
6. Miksovska J., Yom J., Diamond B., Larsen R.W. Spectroscopic and photothermal study of myoglobin conformational changes in the presence of sodium dodecyl sulfate // Biomacromolecules. 2006. V. 7. № 2. P. 476–482.
7. Zhang Y., Stern E.A. Xafs study of co binding to denatured cytochrome c // Physica B: Condensed Matter. 1995. V. 208–209. P. 727–728.
8. Jung C., Ristau O., Schulze H., Sligar S.G. The co stretching mode infrared spectrum of substrate‐free cytochrome p ‐450cam—co // Eur. J. Biochem. 1996. V. 235. № 3. P. 660–669.
9. Cassina A.M., Hodara R., Souza J.M., Thomson L., Castro L., Ischiropoulos H., Freeman B.A., Radi R. Cytochrome c nitration by peroxynitrite // J. Biol. Chem. 2000. V. 275. № 28. P. 21409–21415.
10. Basova L.V., Kurnikov I.V., Wang L., Ritov V.B., Belikova N.A., Vlasova I.I., Pacheco A.A., Winnica D.E., Peterson J., Bayir H., Waldeck D.H., Kagan V.E. Cardiolipin switch in mitochondria:  Shutting off the reduction of cytochrome c and turning on the peroxidase activity // Biochemistry. 2007. V. 46. № 11. P. 3423–3434.
11. Kapetanaki S.M., Silkstone G., Husu I., Liebl U., Wilson M.T., Vos M.H. Interaction of carbon monoxide with the apoptosis-inducing cytochrome ccardiolipin complex // Biochemistry. 2009. V. 48. № 7. P. 1613–1619.
12. Battistuzzi G., Borsari M., Rossi G., Sola M. Effects of solvent on the redox properties of cytochrome c: Cyclic voltammetry and 1h nmr experiments in mixed water-dimethylsulfoxide solutions // Inorganica Chimica Acta. 1998. V. 272. № 1. P. 168–175.
13. Larsen R.W. Ligand binding subsequent to co photolysis of methionine-modified cytochrome c // Biochimica et Biophysica Acta (BBA) – General Subjects. 2003. V. 1619. № 1. P. 15–22.
14. Appaix F., Minatchy M.-N., Riva-Lavieille C., Olivares J., Antonsson B., Saks V.A. Rapid spectrophotometric method for quantitation of cytochrome c release from isolated mitochondria or permeabilized cells revisited // Biochim. Biophys. Acta. 2000. V. 1457. № 3. P. 175–181.
15. Rosell F.I., Mauk A.G. Spectroscopic properties of a mitochondrial cytochrome c with a single thioether bond to the heme prosthetic group // Biochemistry. 2002. V. 41. № 24. P. 7811–7818.
16. Borisenko G.G., Osipov A.N., Kazarinov K.D., Vladimirov YU.A. Nitrozil'nye kompleksy citohromov mitohondrial'noj cepi – pervichnye hromofory v mekhanizme fotoaktivacii dyhaniya // Biologicheskie Membrany. 2002. T. 19. № 5. S. 378–390.
17. Tuominen E.K.J., Wallace C.J.A., Kinnunen P.K.J. Phospholipid-cytochrome c interaction: Evidence for the extended lipid anchorage // J. Biol. Chem. 2002. V. 277. № 11. P. 8822–8826.
18. Macyk J., Eldik R.v. Electron transfer reaction between cytochrome c and trisoxalatocobalt(III) // J. Chem. Soc., Dalton Trans. 2001. № 15. P. 2288–2292.
19. Nantes I.L., Faljoni-Alario A., Vercesi A.E., Santos K.E., Bechara E.J.H. Liposome effect on the cytochrome c-catalyzed peroxidation of carbonyl substrates to triplet species // Free Radical Biol. Med. 1998. V. 25. № 4. P. 546–553.
20. Silkstone G., Jasaitis A., Vos M.H., Wilson M.T. Geminate carbon monoxide rebinding to a c-type haem // Dalton Trans. 2005. № 21. P. 3489– 3494.
21. Hagen S.J., Latypov R.F., Dolgikh D.A., Roder H. Rapid intrachain binding of histidine-26 and histidine-33 to heme in unfolded ferrocytochrome c // Biochemistry. 2002. V. 41. № 4. P. 1372–1380.
22. Karpefors M., Wilson M.T., Brzezinski P. Photoinduced electron transfer from carboxymethylated cytochrome c to plastocyanin // Biochim. Biophys. Acta. 1998. V. 1364. № 3. P. 385–389.
23. Mathews A.J., Brittain T. Some reactions of carbon monoxide and oxygen with carbodi-imide-modified cytochrome c // Biochem. J. 1991. V. 276. № 1. P. 121–124.
24. Hollis V.S., Palacios-Callender M., Springett R.J., Delpy D.T., Moncada S. Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy // Biochim. Biophys. Acta. 2003. V. 1607. № 2. P. 191–202.
25. Qiao F., Hu J., Zhu H., Luo X., Zhu L., Zhu D. Site-specific hydrolysis of horse heart cytochrome c and apocytochrome c promoted by palladium(II) complex // Polyhedron. 1999. V. 18. № 11. P. 1629–1633.
26. Noda I. Generalized two-dimensional correlation method applicable to infrared, raman, and other types of spectroscopy. 1993.
27. Czarnecki M.A. Interpretation of two-dimensional correlation spectra: Science or art? // Applied Spectroscopy. 1998. V. 52. № 12. P. 1583–1590.