NEW INSIGHTS INTO THE CHEMISTRY AND FUNCTIONS OF COENZYME Q

Transcription

1 NEW INSIGHTS INT THE CHEMISTRY AND FUNCTINS F CENZYME Q Faculty of Medical Sciences, University Goce Delcev Stip Symposium Biomedicine, Rubin Gulaboski, Valentin Mirceski, Ivan Bogeski, Sasa Mitrev, Kokoskarova Pavlinka Reinhard Kappl, Velo Markovski, Milkica Janeva, Markus Hoth

2 Nobel Prize Chemistry Piter Mitchel 1978 Coenzyme Q10 is a crucial redox mediator in the Mitochondrial Electron Transport Chain-ATP production

3 QUINNE (oxidized form) 3HC 3HC HC 3HC QUINL (reduced form)

4 Reactive xygen Species = Mitochondrial electron transport chain is a major source of highly dangerous RS!!!

5 :..... Superoxide dismutase e -. - e - e -. e - 2 H 2 2 H H : : H:::H.....:H.. H::H.. xygen Superoxide Hydrogen anion radical peroxide Glutathione Peroxidase Catalase Hydroxyl radical Water Structures of the most common Reactive xygen Species evaluated from 2

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7 Effective cure against RS are the Antioxidants Ubiquinol-moderate antioxidant

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10 Number of publications The number of papers on CoQ10 increases permanently Publication year

11 We started working on this project in 2006

12 Effect of Ca 2+ ions to the redox process of 2-palmytoilquinonetransfer of Ca 2+ across lipid membrane Structure of 2-palmytoilhydroquinone This compound can bind and transfer Ca 2+ ions across biomimetic membranes ur Aim: to see whether CoQ s have something to do with Ca 2+ transfer across mitochondrial membranes

13 Why Ca 2+? Ca 2+ -is one of the most important secondary messengers

14 Mechanisms of Ca 2+ transfer in mitochondria???

15 H 3 C n( 2 HC-( 3 HC)C=HC-CH 2 ) H 3 C H 3 C Coenzyme Q 1 Coenzyme Q 10

16 I / A -3E-05 Experiments with Coenzyme Q1-CoQ1 Cyclic voltammogram of 100 µm CoQ1 in ph of E-05-3E E / V

17 I / A -3E E-05 ph = 2.0 (black) ph = 2.7 (blue) ph = 3.5 (green) ph = 4.5, 7.4 and 9.2 (orange, pink and brown) ph increase E / V ph dependence of the redox process of CoQ1-the yellow form in the ph range from 1 to 9

18 H 3 C H 3 C H H 3 C - + 2e + 2H+ H 3 C H Coenzyme Q 1 (oxidized) Coenzyme Q 1 (reduced)

19 I / A -2.4E E-05 CV of 100µM CoQ-1-in ph of 7,00 (yellow native form); insensitivity to Ca 2+ ; c(ca 2+ )/mm = 0 (black); 1 (green); 10 (orange);50 (black) and 100 (rose) + 2e- + 2Ca 2+ =? CoQ1-the yellow form= insensitive to Ca 2+ ions -8E E E / V That is,...end F THE STRY, or MAY BE NT? -1.4

20 I / A I / A I / A I / A minutes I II II' I' 60 minutes II Cyclic voltammograms of CoQ1 recorded in 1 mol/l NaH. Scans recorded after 3 min (1) 1 h (2) 3 h (3) and 24 h (4). Scan rate of 30 mv/s, c(coq1) = mmol/l I minutes I' II' II Significant changes in the voltammetric responses have been observed when CoQ1 was dissolved in 1M NaH! I I' II' hours II II' E / V

21 TASK: What kind of reaction goes on? what are the products and which are the chemical features of the products, what is the mechanism...we explored plenty of techniques and methods

22 UV-Vis spectrum of 10 µm CoQ1 recorded in kinetic mode in 1 M NaH

23 I / A -3E-05-2E-05-1E µmol/l CoQ-1 in ph of 7. Black curve-yellow form of CoQ1 Red curve-coq1 firstly dissolved in NaH 0 1E E E / V Comparison of the cyclic voltammograms of CoQ1 directly dissolved in ph of 7.00, and of CoQ1 initially dissolved in 1 M NaH for 45 min and retitrated afterwards to ph of

24 ph increase Effect of ph to the redox processes of CoQ1. In this situation CoQ1 was in contact with 0.1 M NaH for 60 minutes Next logical task: DETERMINE the MECHANISM and the STRUCTURE of the New Benzoquinone Product

25 Electron Paramagnetic resonance-epr-suitable technique for structure evaluation by the radical species (many quinones form radicals when dissolved in alkaline media) Simulated EPR spectrum of the radical

26 EPR spectrum of CoQ1 after reduction with half-equimolar amount of NaBH 4 in ph of 7.00 EPR spectrum of CoQ1 obtained in 0.1 M NaH but without using any reductant!! CH 2 Nine-line EPR spectrum od parent CoQ1 corresponds to presence of one and one CH 2 group in the structure

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29 NEXT STEP to determine the structure-nmr EXPERIMENTS NMR spectrum of CoQ1 in D 2 Protons of -

30 Protons of - CoQ1-5 minutes in NaD retitrated afterwards to pd of 7.00 Signal from the protons of METHANL

31 Protons of - CoQ1-10 minutes in NaD Retitrated afterwards to pd of 7.00 Signal from the protons of METHANL

32 Protons of - CoQ1-25 minutes in NaD Retitrated afterwards to pd of 7.00 Signal from the protons of METHANL

33 Protons of - CoQ1-60 minutes in NaD Retitrated afterwards to pd of 7.00 Signal from the protons of METHANL

34 HPLC MS Spectra of Coenzyme Q1 Recorded in ph of 7.00 In presence of Cytocrome P450 r after reaction with NaH for 60 minutes

35 Where METHANL does come from, when CoQ is dissolved in alkaline media? -from the methyl group? or -from the METHXY - group? By using methyl benzoquinone derivatives we found that the methyl group CAN NT BE CLEAVED from the aromatic ring!!! 2-methyl-benzoquinone Tetramethyl benzoquinone (duroquinone)

36 H 3 C H 3 C + 2H - H 3 C H 3 C H Coenzyme Q 1 -demethylated Coenzyme Q 1 (2,3-dihydroxo-5-methyl-6-isoprenyl-benzoquinone)

37 Ratio of the signal of Methanol vs Methoxy groups from NMR experiments of CoQ1 in NaD

38 New product is able to bind Ca 2+ ions in stochiometric ratio 1:2 (L:M 2+ ) concentration of Ca 2+ increase New form of CoQ-1, sensitivity to Ca 2+ ions

39 The slope of 59mV indicates a complex of a type 1:2 (L to M) Dependence of the mid-peak potential of the cyclic voltammograms of new form of CoQ1 on the logarithm of Ca 2+ concentration

40 H 3 C H 3 C + 2H - H 3 C H 3 C H Coenzyme Q 1 -demethylated Coenzyme Q 1

41 EXPERIMENTS with Coenzyme Q10-CoQ10 Native CoQ10 is absolutely insoluble in water, but... it can be dissolved in NaH...and it can be transformed into a new form in presence of H - anions present in the organic phase

42 Coenyzme Q10 in presence of 1 M NaH 5 µm Coenyzme Q10 in presence of 1 M NaH and retitrated to ph of 7.0 after 3 weeks The membrane-bound enzyme Cytochrome P450 does the same task as NaH to CoQ10!!!

43 Effect of Ca 2+ to the voltammetric response of CoQ10 in presence of CYP450 in ph of 7.40

44 - H 3 C + 2H - + H 3 C - R CYP450 R - + H 2 H H - 2CH3 H 2. Stabilization of the -cleaved CoQ10 products in water - R 3. Reduction of the -cleaved CoQ10 product and binding of Ca 2+ - H H R Ca e - - R Ca 2+ - R Ca 2+ R -

45 Experiments with CoQ10 embedded in organic membrane in presence of rganic Hydroxide to show whether the new form of CoQ10 can transfer Ca 2+ ions across biological membranes Ca 2+ out Lipid membrane CoQ10 embedded + H - electrode

46 Mitochondrial Ca 2+ influx (slope in %) Ratio (340/380) WT+Ca+Cyt WT+Ca Time (min) 105% 100% *** 95% 90% 85% 80% 75% Mito WT Mito CoQ10 K Experiments with wild types of mitochondria and knockout mitochondria depleted of CoQ10 in absence and in presence of CYTP450. Effect of Ca 2+ ions

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48 Strong Antioxidative Properties of novel Hydroxilated Derivatives of Coenzyme Q

49 Summary NATIVE Coenzyme Q-10 Highly lipophilic, moderate antioxidants, with no potential for metal binding H 3 C n( 2 HC-( 3 HC)C=HC-CH 2 ) H 3 C H 3 C Coenzyme Q 1 Coenzyme Q 10 NEW H-Coenzyme Q-10 Amphiphilic, strong antioxidants, Good potential for metal binding H 3 C H 3 C - H 3 C + 2H - H 3 C H Coenzyme Q 1 -demethylated Coenzyme Q 1

50 Can the story about Mitochondrial Electron Transport Chain be modified in presence of Cyt P-450?

51 SUMMARY Plenty of CoQ family members are present in the living systems The chemistry and most of the functions of the native forms of Coenzyme Q family members are mainly portrayed in the features of the 2e-/2H + redox reaction (electron and proton transfer) that leads to reversible transformation of the quinone to quinol forms. If the Coezyme Q structures are in contact with high concentration of H - ions or CYP450 enzymes, quite different quinonic forms can be obtained. CYP450 and NaH can both induce scission of the both - (methoxy) groups in the structure of the Coenzyme Q family members, thus creating so called demethylated quinones that bear charge of 2-. These new Coenzyme Q structures formed in alkaline media (or in presence of CYP450) are more polar than their parent compounds, while also having much stronger antioxidative features. The inherent properties of the new Coenzyme Q structures to bind the earth-alkaline cations upon their reduction classify these compounds as potential facilitators for transferring of metal ions across biological membranes.

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54 Rubin Gulaboski, Ivan Bogeski, Reinhard Kappl, Markus Hoth, Benzoquinones based Antioxidants, European Patent ffice, Munich 2010, PATENT No

55 Main people involved in this project A. in Homburg B. Saarbrücken I. Bogeski M. Hoth Bernd Morgenstern Barbara Kutzky D: MKD Richi Kohler R. Kappl ACKNWLEDGMENT also to Alexander von Humboldt Foundation V. Markovski C. UNi K Lautern Prof. Hermann Prof. Mirceski

56 Experiments with Coenzyme Q10-CoQ10 Solubility of CoQ10 in water is bellow 10 pm!!! Impossible to perform experiments in water media! CoQ10 studied voltammetrically in Thin-film voltammetry set-up Electron conductor, graphite electrode rganic droplet of org. solution water of an immiscible electroactive compound solvent CoQ e- CoQ CoQ 10 H + H + H + H - H - H + Cat 2+ Cat 2+ 2An - Liquid-Liquid interface

57 Color of solutions of Benzoquinones with 2 H groups in its structure Color of solutions of Benzoquinones with 1 H group in its structure

58 Benzoquinones with 2-H groups are able to bind earth-alkaline cations in ratio 1:2 Benzoquinones with 1-H group are NT able to bind (at least not strongly) earth-alkaline cations

59 I / A -3E-05-2E-05-1E E E E / V

60 -E/V NADH -0.2 _ 0.0 _ CoQ Complex III Complex IV 0.2 _ The processes in the mitochondrial electron transfer chain are driven by the differences in the standard redox potentials of the contributors