Russian Journal of Biological Physics and Chemisrty Russian Journal of Biological Physics and Chemisrty АКТУАЛЬНЫЕ ВОПРОСЫ БИОЛОГИЧЕСКОЙ ФИЗИКИ И ХИМИИ 2499-9962 55022 10.29039/rusjbpc.2022.0522 МЕДИЦИНСКАЯ БИОФИЗИКА И БИОФИЗИЧЕСКАЯ ХИМИЯ MEDICAL BIOPHYSICS AND BIOPHYSICAL CHEMISTRY МЕДИЦИНСКАЯ БИОФИЗИКА И БИОФИЗИЧЕСКАЯ ХИМИЯ CYTOCHROME P450 SYSTEM MAY BE INVOLVED IN THE LIGHT EMISSION OF HIGHER FUNGI Система цитохрома Р450 может участвовать в светоизлучении высших грибов Ронжин Н. О. Ronzhin N. O. roniol@mail.ru Посохина Е. Д. Posokhina E. D. Могильная О. А. Mogilnaya O. A. ol_mog@mail.ru Пузырь А. П. Puzyr A. P. Гительзон И. И. Gitelson J. I. Бондарь В. С. Bondar V. S. ФИЦ КНЦ СО РАН Красноярск Россия FRC KSC SB RAS Krasnoyarsk Russian Federation ФИЦ КНЦ СО РАН Красноярск Россия FRC KSC SB RAS Krasnoyarsk Russian Federation ФИЦ КНЦ СО РАН Красноярск Россия FRC KSC SB RAS Krasnoyarsk Russian Federation ФИЦ КНЦ СО РАН Красноярск Россия FRC KSC SB RAS Krasnoyarsk Russian Federation ФИЦ КНЦ СО РАН Красноярск Россия FRC KSC SB RAS Krasnoyarsk Russian Federation ФИЦ КНЦ СО РАН Красноярск Россия FRC KSC SB RAS Krasnoyarsk Russian Federation ФИЦ КНЦ СО РАН Красноярск Россия FRC KSC SB RAS Krasnoyarsk Russian Federation 25 06 2022 25 06 2022 7 2 320 324 20 06 2022 20 06 2022 https://rusjbpc.ru/en/nauka/article/55022/view

Приводятся данные, свидетельствующие в пользу участия системы цитохрома Р450 в светоизлучении высших грибов. Из мицелия разных видов светящихся базидиомицетов получены экстракты, которые содержат грибные люминесцентные системы, обеспечивающие свечение in vitro. Условия выделения систем (обработка биомассы ультразвуком, осветление гомогенатов центрифугированием при 40000g) указывают на наличие в экстрактах мембранных структур, в частности, микросом, образующихся в результате разрушения эндоплазматического ретикулума (ЭПР) ультразвуком. Дифференциальный спектральный анализ экстрактов, обработанных дитионитом натрия и СО, выявил наличие двух пиков поглощения при 410 нм и 450 нм, что указывает на наличие цитохромов Р450 и b5. Свечение экстрактов стимулируется восстановленными пиридиновыми нуклеотидами, однако установлено, что больший уровень свечения наблюдается при добавках НАДФН, по сравнению с НАДН. Значения Vmax световой эмиссии под действием НАДФН практически в 2 раза выше, а значения кажущихся Кm в 2 раза ниже, по сравнению с НАДН. Добавки пероксида водорода значительно (от нескольких раз до 1-2 порядков) увеличивают интенсивность свечения экстрактов, активированных НАД(Ф)Н. Установлено, что добавки флуконазола (5-60 мкг в пробе) существенно ингибируют световую эмиссию экстрактов – наблюдается снижение как исходного уровня свечения, так и свечения после добавок НАД(Ф)Н. Совокупность полученных данных свидетельствует, что в механизме светоизлучения высших грибов может участвовать ассоциированная с мембранами ЭПР система цитохрома Р450 с вовлечением в процесс электронно-транспортных ферментных систем: НАДФН-зависимая редуктаза цитохрома Р450 – цитохром Р450 и НАДН-зависимая редуктаза цитохрома b5 – цитохром b5 – цитохром Р450. В этом случае цитохром Р450 может осуществлять гидроксилирование гиспидина (прекурсор субстрата люминесцентной реакции) с образованием люциферина и катализировать его окисление в присутствии АФК с излучением света.

The paper presents data that testify in favor of the participation of the cytochrome P450 system in the light emission of higher fungi. Extracts from mycelia of different species of luminous basidiomycetes containing fungal luminescent systems that provide luminescence in vitro were obtained. Applied conditions for the isolation of luminescent systems (sonication, centrifugation at 40000g) indicate the presence of membrane structures in the extracts, in particular, microsomes formed as a result of ultrasonic disintegration of the endoplasmic reticulum (ER). Differential spectral analysis of the extracts revealed the presence of two absorption peaks at 410 nm and 450 nm, which indicates the presence of cytochromes b5 and P450. The luminescence of the extracts is stimulated by reduced pyridine nucleotides, however, the addition of NADPH causes a higher level of luminescence compared with NADH. The addition of hydrogen peroxide significantly (from several times to 1-2 orders of magnitude) increases the luminescence intensity of extracts activated by NAD(P)H. The addition of fluconazole significantly inhibits the light emission of extracts. The data obtained indicates that the cytochrome P450 system associated with ER membranes may participate in the mechanism of light emission of higher fungi with the involvement in the process of electron transport enzyme systems: NADPH-dependent reductase of cytochrome P450 - cytochrome P450 and NADH-dependent reductase of cytochrome b5 - cytochrome b5 - cytochrome P450. In this case, cytochrome P450 may hydroxylate hispidin (precursor of the luminescent reaction substrate) to form luciferin and catalyze its oxidation in the presence of ROS with light emission.

 светящиеся высшие грибы мицелий гиспидин система цитохрома Р450 восстановленные пиридиновые нуклеотиды флуконазол пероксид водорода luminous higher fungi mycelium hispidin cytochrome P450 system reduced pyridine nucleotides fluconazole hydrogen peroxide Исследования выполнены в рамках государственного задания Министерства науки и высшего образования РФ (проект № 0287-2021-0016).

Bondar V.S., Puzyr A.P., Purtov K.V., Petunin A.I., Burov A.E., Rodicheva E.K., Medvedeva S.E., Shpak B.A., Tyaglik A.B., Shimomura O., Gitelson J.I. Isolation of luminescence system from the luminescent fungus Neonothopanus nambi. Doklady Biochemistry and Biophysics, 2014, vol. 455, doi: 10.1134/S1607672914020045. Bondar V.S., Puzyr A.P., Purtov K.V., Petunin A.I., Burov A.E., Rodicheva E.K., Medvedeva S.E., Shpak B.A., Tyaglik A.B., Shimomura O., Gitelson J.I. Isolation of luminescence system from the luminescent fungus Neonothopanus nambi. Doklady Biochemistry and Biophysics, 2014, vol. 455, doi: 10.1134/S1607672914020045. Purtov K.V., Petushkov V.N., Baranov M.S. et al. The chemical basis of fungal bioluminescence. Angewandte Chemie International Edition, 2015, vol. 54, doi: 10.1002/anie.201501779. Purtov K.V., Petushkov V.N., Baranov M.S. et al. The chemical basis of fungal bioluminescence. Angewandte Chemie International Edition, 2015, vol. 54, doi: 10.1002/anie.201501779. Oba Y., Suzuki Y., Martins G.N.R. et al. Identification of hispidin as a bioluminescent active compound and its recycling biosynthesis in the luminous fungal fruiting body. Photochemical & Photobiological Sciences, 2017, vol. 16, doi: 10.1039/c7pp00216e. Oba Y., Suzuki Y., Martins G.N.R. et al. Identification of hispidin as a bioluminescent active compound and its recycling biosynthesis in the luminous fungal fruiting body. Photochemical & Photobiological Sciences, 2017, vol. 16, doi: 10.1039/c7pp00216e. Kaskova Z.M., Dorr F.A., Petushkov V.N. et al. Mechanism and color modulation of fungal bioluminescence. Science Advances, 2017, vol. 3, doi: 10.1126/sciadv.1602847. Kaskova Z.M., Dorr F.A., Petushkov V.N. et al. Mechanism and color modulation of fungal bioluminescence. Science Advances, 2017, vol. 3, doi: 10.1126/sciadv.1602847. Puzyr A.P. Medvedeva S.E., Artemenko K.S., Bondar V.S. Luminescence of cold extracts from mycelium of luminous basidiomycetes during long-term storage. Current Research in Environmental & Applied Mycology, 2017, vol. 7, doi: 10.5943/cream/7/3/9. Puzyr A.P. Medvedeva S.E., Artemenko K.S., Bondar V.S. Luminescence of cold extracts from mycelium of luminous basidiomycetes during long-term storage. Current Research in Environmental & Applied Mycology, 2017, vol. 7, doi: 10.5943/cream/7/3/9. Kotlobay A.A., Sarkisyan K.S., Mokrushina Y.A. et al. Genetically encodable bioluminescent system from fungi. Proceedings of the National Academy of Sciences, 2018, vol. 115, doi: 10.1073/pnas.1803615115. Kotlobay A.A., Sarkisyan K.S., Mokrushina Y.A. et al. Genetically encodable bioluminescent system from fungi. Proceedings of the National Academy of Sciences, 2018, vol. 115, doi: 10.1073/pnas.1803615115. Teranishi K. Bioluminescence and chemiluminescence abilities of trans-3-hydroxyhispidin on the luminous fungus Mycena chlorophos. Luminescence, 2018, vol. 33, doi: 10.1002/bio.3540. Teranishi K. Bioluminescence and chemiluminescence abilities of trans-3-hydroxyhispidin on the luminous fungus Mycena chlorophos. Luminescence, 2018, vol. 33, doi: 10.1002/bio.3540. Puzyr A.P., Burov A.E., Medvedeva S.E., Burova O.G., Bondar V.S. Two forms of substrate for the bioluminescent reaction in three species of basidiomycetes. Mycology, 2019, vol. 10, doi: 10.1080/21501203.2019.1583688. Puzyr A.P., Burov A.E., Medvedeva S.E., Burova O.G., Bondar V.S. Two forms of substrate for the bioluminescent reaction in three species of basidiomycetes. Mycology, 2019, vol. 10, doi: 10.1080/21501203.2019.1583688. Garcia-Iriepa C., Losantos R., Fernandez-Martinez D., Sampedro D., Navizet I. Fungal light emitter: understanding its chemical nature and pH-dependent emission in water solution. The Journal of Organic Chemistry, 2020, vol. 85, doi: 10.1021/acs.joc.0c00246. Garcia-Iriepa C., Losantos R., Fernandez-Martinez D., Sampedro D., Navizet I. Fungal light emitter: understanding its chemical nature and pH-dependent emission in water solution. The Journal of Organic Chemistry, 2020, vol. 85, doi: 10.1021/acs.joc.0c00246. Ronzhin N.O., Posokhina E.D., Mogilnaya O.A., Bondar V.S. Finding the light emission stimulator of Neonothopanus nambi basidiomycete and studying its properties. Doklady Biochemistry and Biophysics, 2022, vol. 503, doi: 10.1134/S1607672922020120. Ronzhin N.O., Posokhina E.D., Mogilnaya O.A., Bondar V.S. Finding the light emission stimulator of Neonothopanus nambi basidiomycete and studying its properties. Doklady Biochemistry and Biophysics, 2022, vol. 503, doi: 10.1134/S1607672922020120. Shimomura O. Superoxide-triggered chemiluminescence of the extract of luminous mushroom Panellus stipticus after treatment with methylamine. Journal of Experimental Botany, 1991, vol. 42, no. 237, pp. 555-560. Shimomura O. Superoxide-triggered chemiluminescence of the extract of luminous mushroom Panellus stipticus after treatment with methylamine. Journal of Experimental Botany, 1991, vol. 42, no. 237, pp. 555-560. Shimomura O. The role of superoxide dismutase in regulating the light emission of luminescent fungi. Journal of Experimental Botany, 1992, vol. 43, no. 256, pp. 1519-1525. Shimomura O. The role of superoxide dismutase in regulating the light emission of luminescent fungi. Journal of Experimental Botany, 1992, vol. 43, no. 256, pp. 1519-1525. Shimomura O. Bioluminescence: chemical principles and methods. Singapore: World Scientific Publishing Co. Pte. Ltd., 2006, 470 p. Shimomura O. Bioluminescence: chemical principles and methods. Singapore: World Scientific Publishing Co. Pte. Ltd., 2006, 470 p. Bondar V.S., Puzyr A.P., Purtov K.V., Medvedeva S.E., Rodicheva E.K., Gitelson J.I. The luminescent system of the luminous fungus Neonothopanus nambi. Doklady Biochemistry and Biophysics, 2011, vol. 438, doi: 10.1134/S1607672911030082. Bondar V.S., Puzyr A.P., Purtov K.V., Medvedeva S.E., Rodicheva E.K., Gitelson J.I. The luminescent system of the luminous fungus Neonothopanus nambi. Doklady Biochemistry and Biophysics, 2011, vol. 438, doi: 10.1134/S1607672911030082. Bondar V.S., Shimomura O., Gitelson J.I. Luminescence of higher mushrooms. Journal of Siberian Federal University. Biology, 2012, vol. 4, no. 5, pp. 331-351. Bondar V.S., Shimomura O., Gitelson J.I. Luminescence of higher mushrooms. Journal of Siberian Federal University. Biology, 2012, vol. 4, no. 5, pp. 331-351. Bondar V.S., Rodicheva E.K., Medvedeva S.E., Tyulkova N.A., Tyaglik A.B., Shpak B.A., Gitelson J.I. On the mechanism of luminescence of the fungus Neonothopanus nambi. Doklady Biochemistry and Biophysics, 2013, vol. 449, DOI: 10.1134/S1607672913020075. Bondar V.S., Rodicheva E.K., Medvedeva S.E., Tyulkova N.A., Tyaglik A.B., Shpak B.A., Gitelson J.I. On the mechanism of luminescence of the fungus Neonothopanus nambi. Doklady Biochemistry and Biophysics, 2013, vol. 449, DOI: 10.1134/S1607672913020075. Kobzeva T.V., Melnikov A.R., Karogodina T.Y., Zikirin S.B., Stass D.V., Molin Yu.N., Rodicheva E.K., Medvedeva S.E., Puzyr A.P., Burov A.E., Bondar V.S., Gitelson J.I. Stimulation of luminescence of mycelium of luminous fungus Neonothopanus nambi by ionizing radiation. Luminescence, 2014, vol. 29, doi: 10.1002/bio.2656. Kobzeva T.V., Melnikov A.R., Karogodina T.Y., Zikirin S.B., Stass D.V., Molin Yu.N., Rodicheva E.K., Medvedeva S.E., Puzyr A.P., Burov A.E., Bondar V.S., Gitelson J.I. Stimulation of luminescence of mycelium of luminous fungus Neonothopanus nambi by ionizing radiation. Luminescence, 2014, vol. 29, doi: 10.1002/bio.2656. Mogilnaya O.A., Ronzhin N.O., Artemenko K.S., Bondar V.S. Morphological properties and levels of extracellular peroxidase activity and light emission of the basidiomycete Armillaria borealis treated with β-glucosidase and chitinase. Mycosphere, 2017, vol. 8, doi: 10.5943/mycosphere/8/4/11. Mogilnaya O.A., Ronzhin N.O., Artemenko K.S., Bondar V.S. Morphological properties and levels of extracellular peroxidase activity and light emission of the basidiomycete Armillaria borealis treated with β-glucosidase and chitinase. Mycosphere, 2017, vol. 8, doi: 10.5943/mycosphere/8/4/11. Mogilnaya O.A., Ronzhin N.O., Bondar V.S. Estimating levels of light emission and extracellular peroxidase activity of mycelium of luminous fungus Neonothopanus nambi treated with β-glucosidase. Current Research in Environmental & Applied Mycology, 2018, vol. 8, doi: 10.5943/cream/8/1/6. Mogilnaya O.A., Ronzhin N.O., Bondar V.S. Estimating levels of light emission and extracellular peroxidase activity of mycelium of luminous fungus Neonothopanus nambi treated with β-glucosidase. Current Research in Environmental & Applied Mycology, 2018, vol. 8, doi: 10.5943/cream/8/1/6. Airth R.L., McElroy W.D. Light emission from extracts of luminous fungi. Journal of Bacteriology, 1959, vol. 77, doi: 10.1128/jb.77.2.249-250.1959. Airth R.L., McElroy W.D. Light emission from extracts of luminous fungi. Journal of Bacteriology, 1959, vol. 77, doi: 10.1128/jb.77.2.249-250.1959. Airth R.L. Foerster G.E. The isolation of catalytic components required for cell-free fungal bioluminescence. Archives of Biochemistry and Biophysics, 1962, vol. 97, doi: 10.1016/0003-9861(62)90124-8. Airth R.L. Foerster G.E. The isolation of catalytic components required for cell-free fungal bioluminescence. Archives of Biochemistry and Biophysics, 1962, vol. 97, doi: 10.1016/0003-9861(62)90124-8. Omura T., Sato R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. The Journal of Biological Chemistry, 1964, vol. 239, no. 7, pp. 2370-2378. Omura T., Sato R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. The Journal of Biological Chemistry, 1964, vol. 239, no. 7, pp. 2370-2378. Peisach J., Stern J.O., Blumberd W.E. 1973 - Optical and magnetic probes of the structure of cytochrome P-450's. Drug Metabolism and Disposition, vol. 1, no. 1, pp. 45-61. Peisach J., Stern J.O., Blumberd W.E. 1973 - Optical and magnetic probes of the structure of cytochrome P-450's. Drug Metabolism and Disposition, vol. 1, no. 1, pp. 45-61. Moskaleva N.E., Zgoda V.G. Modern methods of cytochrome P450 analysis. Biochemistry (Moscow) Supplement. Series B: Biomedical Chemistry, 2013, vol. 7, doi: 10.1134/S1990750813020078. Moskaleva N.E., Zgoda V.G. Modern methods of cytochrome P450 analysis. Biochemistry (Moscow) Supplement. Series B: Biomedical Chemistry, 2013, vol. 7, doi: 10.1134/S1990750813020078. 8. Kelly S.L. et al. An old activity in the cytochrome P450 superfamily (CYP51) and a new story of drugs and resistance. Biochemical Society Transaction, 2001, vol. 29, doi: 10.1042/0300-5127:0290122. 8. Kelly S.L. et al. An old activity in the cytochrome P450 superfamily (CYP51) and a new story of drugs and resistance. Biochemical Society Transaction, 2001, vol. 29, doi: 10.1042/0300-5127:0290122. Maddy A.H. Biochemical analysis of membranes. London: Chapman & Hall Ltd., 1976, 513 p. Maddy A.H. Biochemical analysis of membranes. London: Chapman & Hall Ltd., 1976, 513 p. Archakov A.I., Bachmanova G.I. Cytochrome P450 and active oxygen. London: Taylor & Francis, 1990, 339 p. Archakov A.I., Bachmanova G.I. Cytochrome P450 and active oxygen. London: Taylor & Francis, 1990, 339 p. Lewis D.F.V. Guide to cytochromes P450. Structure and function. London, New York: Taylor & Francis, 2001, 215 p. Lewis D.F.V. Guide to cytochromes P450. Structure and function. London, New York: Taylor & Francis, 2001, 215 p. Munro A.W., McLean K.J., Grant J.L., Makris T.M. Structure and function of the cytochrome P450 peroxygenase enzymes. Biochemical Society Transactions, 2018, vol. 46, doi: 10.1042/BST20170218. Munro A.W., McLean K.J., Grant J.L., Makris T.M. Structure and function of the cytochrome P450 peroxygenase enzymes. Biochemical Society Transactions, 2018, vol. 46, doi: 10.1042/BST20170218.