Lecture 2 on P450s for Bb - PDF

Cytochromes P450 flurbiprofen heme BIO-5002A Biochemistry, Professor Julea Butt ([email protected]) Lecture Outline Last Week:...

Cytochromes P450 flurbiprofen heme BIO-5002A Biochemistry, Professor Julea Butt ([email protected]) Lecture Outline Last Week: PDB file 1R9O  Introduction to Cytochrome P450 enzymes  Metabolism of Endogenous and Exogenous Molecules  Induction and Inhibition of Cytochromes P450 Today:  The catalytic mechanism of cytochromes P450  Cytochromes P450 R3CH + O2 + NADPH + H+  R3COH + NADP+ + H2O where R3CH is a hydrophobic / lipophilic compound. NADPH NADP+ NADPH NADP+ Ferredoxin Reductase FAD e- NADPH Cytochrome FAD P450 Oxidoreductase Ferredoxin FeS matrix e- FMN cytosol inner heme heme mitochondrial ER membrane R3CH R3COH membrane R3CH R3COH O2 H2 O O2 H2O Cytochrome P450 Cytochrome P450 Class I Class II Understanding the Catalytic Cycle of Cytochromes P450 R3CH + O2 + NADPH + H+  R3COH + NADP+ + H2O where R3CH is a hydrophobic / lipophilic compound. Considering the overall transformation catalysed by cytochromes P450 raises a number of questions about the order of events that occur in the catalytic cycle:  Must one substrate bind before the others?  Where are the substrates bound within the active site?  What is the role of the heme cofactor in catalysis?  Are the products released simultaneously or in a defined order? It turns out catalysis by most cytochromes P450 occurs is a precisely ordered sequence of events in order to minimise unwanted side-reactions of the very reactive reduced oxygen species that are intermediates in the reaction pathway.  The Catalytic Mechanism Common to Most Cytochromes P450 R3C-OH R3C-H Fe(III) oxidised R3C-H R3C-H O Fe(III) Fe(II) oxidised reduced H2O e- e- , H + R3C-H R3C-H O2 Fe(II) Fe(II) reduced reduced O2 e- come from NADPH via Ferredoxin Reductase/Ferredoxin (Class I enzymes) or NADPH Cytochrome P450 oxidoreductase (Class II enzymes). Experimental Evidence for the Catalytic Mechanism of Cytochromes P450 The mechanism of cytochromes P450 has been revealed by numerous experiments designed to explore the role of key biochemical concepts; specifically with regard to reaction kinetics, binding constants and redox chemistry. The first steps in the catalytic cycle have been revealed by UV-visible absorbance spectroscopy. Today’s Lecture Content UV-visible absorbance spectroscopy: - identifies the heme cofactor in cytochromes P450 - quantifies the redox activity of cytochromes P450 - demonstrates that a diatomic gas can bind to reduced cytochromes P450 taken together the results above show that: R3CH binding precedes reduction of the heme in the catalytic cycle of cytochromes P450 , and that O2 binds to the reduced heme Learning Outcomes After attending the lectures and pursuing private study that involves reading of relevant text-books, on-line resources and peer-reviewed literature, you will be able to: a) demonstrate understanding of the role of UV-vis spectroscopy in: - identifying the heme cofactor in cytochromes P450 - quantifying the redox activity of cytochromes P450 - demonstrating that a diatomic gas can bind to reduced cytochromes P450 b) describe the mechanism common to most cytochromes P450 and illustrate how information from UV-vis spectroscopy lends support to the sequence of events in the catalytic cycle. References can be found in the slides for the first lecture. Cytochromes P450: Colour and UV-vis Spectroscopy Visible Wavelengths 100 Electronic Absorption Fe(III) 80  (mM-1cm-1) 60 40 20 0 300 400 500 600 700 Wavelength (nm) Electronic Excited State (*) Relative energy N N N N Fe Fe N N N N heme b Electronic (protoporphyrin IX) Ground State () COOH COOH UV-vis Spectrum of Oxidised Fe(III) Cytochromes P450 oxidised 0.6 Absorbance 0.4 0.2 0.0 400 500 600 700 Wavelength (nm)  Molecular Identification by UV-vis Spectroscopy 0.6 Absorbance 0.4 0.2 0.0 400 500 600 700 Wavelength (nm) Cytochrome P450 Azurin – A ‘blue’ copper protein NADP+ NADPH [Fe-S] cluster containing ferredoxin co-substrate identification UV-vis Spectra: Oxidised and Reduced Cytochrome P450 oxidised 0.6 reduced Visible Wavelengths in air +dithionite Absorbance 0.4 0.2 0.0 400 500 600 700 Wavelength (nm) The protein was exposed to the chemical reductant, sodium dithionite Eo’ -500 mV, to generate the sample producing the spectrum of reduced protein above.  Spectroscopy has identified the cofactor in cytochrome P450. Changes in the spectrum on addition of a chemical reductant, sodium dithionite, indicate the cofactor is redox active. Spectroscopy can also be used to define the parameters that quantify the redox activity. Redox Chemistry, the Nernst Equation and Reducing Power Oxidation Is Loss of electrons Reduction Is Gain of electrons } OILRIG electron stoichiometry of the reaction (n) The Half-Reaction reduction of X X + ne- Xn- oxidation of Xn- oxidised form of X reduced form of X Redox Chemistry, the Nernst Equation and Reducing Power Oxidation Is Loss of electrons Reduction Is Gain of electrons } OILRIG electron stoichiometry of the reaction (n) reduction of X X + ne- Xn- oxidation of Xn- oxidised form of X reduced form of X The potential at which 50% of molecules are oxidised and 50% of molecules are reduced is defined as the electrode potential, or, reduction potential for the half reaction. It is given the symbol Eo’ at pH 7 and has units of Volts. Half-reaction Eo' (V) O2 + 4e- + 4H+  2H2O 0.816 the more NO + 2e + 2H  NO + H2O 3 - - + 2 - 0.421 positive the reduction Fe(CN)63- + e-  Fe(CN)64- 0.365 potential the greater Cytochrome c (Fe3+) + e-  Cytochrome c (Fe2+) 0.254 the affinity of the Q + 2H+ + 2e-  QH2 0.045 oxidised FAD + 2e- + 2H+  FADH2 -0.219 molecule for NAD+ + 2e- + H+  NADH -0.320 electrons NADP+ + 2e- + H+  NADPH -0.324  strongest oxidant or 'oxidising agent' Half-reaction Eo' (V) O2 + 4e- + 4H+  2H2O 0.816 the more NO + 2e + 2H  NO + H2O 3 - - + 2 - 0.421 positive the reduction Fe(CN)63- + e-  Fe(CN)64- 0.365 potential the greater Cytochrome c (Fe3+) + e-  Cytochrome c (Fe2+) 0.254 the affinity of the Q + 2H+ + 2e-  QH2 0.045 oxidised FAD + 2e- + 2H+  FADH2 -0.219 molecule for NAD+ + 2e- + H+  NADH -0.320 electrons NADP+ + 2e- + H+  NADPH -0.324  strongest oxidant or 'oxidising agent' Half-reaction Eo' (V) O2 + 4e- + 4H+  2H2O 0.816 the more NO + 2e + 2H  NO + H2O 3 - - + 2 - 0.421 positive the reduction Fe(CN)63- + e-  Fe(CN)64- 0.365 potential the greater Cytochrome c (Fe3+) + e-  Cytochrome c (Fe2+) 0.254 the affinity of the Q + 2H+ + 2e-  QH2 0.045 oxidised FAD + 2e- + 2H+  FADH2 -0.219 molecule for NAD+ + 2e- + H+  NADH -0.320 electrons NADP+ + 2e- + H+  NADPH -0.324 strongest reductant or 'reducing agent' Using the Nernst Equation to Measure Reduction Potentials (Eo’) - Part 1 The Nernst Equation Mass Balance [oxidised] + [reduced] = 100% Eoo ( O x / Re d ) 100 E’ % Mo le c ule s 50 Re d uc e d ( Re d ) 0 100 % Mo le c ule s 50 O x id is e d (O x ) 0 inc r e a s ing ly ne g a t ive inc r e a s ing ly po s it ive E t e nt ial(Volts) Po sample ( Vo lt s ) Using the Nernst Equation to Measure Reduction Potentials (Eo’) - Part 2 syringe containing reductant Esample Voltmeter Air tight seal to avoid introduction of oxygen. light path Potentiometric Titration cuvette Using the Nernst Equation to Measure Reduction Potentials (Eo’) - Part 2 a) Equilibrate an anaerobic sample at defined potential and measure [oxidised] or [reduced] with an appropriate spectroscopy. A typical set up to use UV-vis spectroscopy to monitor oxidation state is shown on the syringe right. containing b) Add an aliquot of reductant via the syringe. Allow the reductant sample to equilibrate at the new potential, record E sample Esample and measure the spectrum of the sample. c) Repeat b) until the sample is fully reduced. Voltmeter d) Convert absorbance at a defined potential to % oxidised molecules and plot % versus Esample (see below). e) Use a computer to fit the data points to the equation from the previous slide and this will tell you E o’ and n (see below). Air tight seal to avoid introduction of oxygen. light path Potentiometric Titration cuvette Using the Nernst Equation to Measure Reduction Potentials (Eo’) - Part 2 a) Equilibrate an anaerobic sample at defined potential and measure [oxidised] or [reduced] with an appropriate spectroscopy. A typical set up to use UV-vis spectroscopy to monitor oxidation state is shown on the syringe right. containing b) Add an aliquot of reductant via the syringe. Allow the reductant sample to equilibrate at the new potential, record E sample Esample and measure the spectrum of the sample. c) Repeat b) until the sample is fully reduced. Voltmeter d) Convert absorbance at a defined potential to % oxidised molecules and plot % versus Esample (see below). e) Use a computer to fit the data points to the equation from the previous slide and this will tell you E o’ and n (see below). Eo ( O x / Re d ) Air tight seal 100 to avoid % introduction Mo le c u le s 50 Re d u c e d of oxygen. ( Re d ) 0 100 Red = data % Mo le c u le s 50 Blue= computer fit light O x id is e d (O x ) path 0 in c r e a s in g ly n e g a t ive in c r e a s in g ly p o s it ive Po t e n t i a l ( Vo lt s ) cuvette Substrate Binding Alters the Reduction Potential of P450 Aliquots of the chemical reductant, sodium dithionite Eo’ -500 mV, used to reduce the enzyme. In one experiment no organic substrate is present (dashed blue line on right), in one experiment an organic substrate is present (continuous blue line on right). Cytochrome P450 0.6 reduced 100 oxidised [oxidised P450] % 0.4 Absorbance 50 0.2 0 -0.7 -0.5 -0.3 -0.1 0.1 0.0 Sample Potential (V) 400 500 600 700 Wavelength (nm) Biochemistry (1997) 36 13816 Substrate Binding Alters the Reduction Potential of P450 Aliquots of the chemical reductant, sodium dithionite Eo’ -500 mV, used to reduce the enzyme. In one experiment no organic substrate is present (dashed blue line on right), in one experiment an organic substrate is present (continuous blue line on right). Cytochrome P450 0.6 reduced 100 oxidised [oxidised P450] % 0.4 Absorbance 50 0.2 0 -0.7 -0.5 -0.3 -0.1 0.1 0.0 Sample Potential (V) 400 500 600 700 Wavelength (nm) Addition of organic substrate raises the Biochemistry (1997) 36 13816 reduction potential of P450. R3CH Binding to P450 Allows Heme Reduction by NADPH Eo' Electron transfer from NADPH to P450 Fe(III) is not possible because Eo’ of NADP+/NADPH is more positive than than of P450 Fe(III)/(II). -200 mV -300 mV NADP+/NADPH P450 Fe(III)/Fe(II) -400 mV Proc Nat Acad Sci (1976) 73 1078 Biochemistry (1997) 36 13816 R3CH Binding to P450 Allows Heme Reduction by NADPH Eo' -200 mV R3CH Bound P450 Fe(III)/Fe(II) -300 mV R3CH NADP /NADPH + P450 Fe(III)/Fe(II) -400 mV R3CH binding to P450 Fe(III) raises Eo’ of the P450 Fe(III)/(II) couple such that it is now less negative than for NADP+/NADPH. NADPH can now reduce the P450 and electron transfer occurs. The Catalytic Mechanism Common to Most Cytochromes P450 R3C-OH R3C-H Fe(III) oxidised R3C-H R3C-H O Fe(III) The redox analysis allows Fe(II) us the understand the first oxidised reduced two steps in the reaction cycle. H2O e- e- , H + R3C-H R3C-H O2 Fe(II) Fe(II) reduced reduced O2 e- come from NADPH via Ferredoxin Reductase/Ferredoxin (Class I enzymes) or NADPH via Cytochrome P450 oxidoreductase (Class II enzymes).  Which wavelength would be best analysed to quantify the extent of P450 reduction? A 0.6 0.4 Absorbance B C 0.2 D 0.0 400 500 600 700 Wavelength (nm) The Catalytic Mechanism Common to Most Cytochromes P450 R3C-OH R3C-H Fe(III) oxidised R3C-H R3C-H O2 binds only after organic Fe(III) O Fe(II) substrate and an e- are oxidised present in the active site. reduced This ensures no reactive H2O oxygen species can be e- formed. e- , H + R3C-H R3C-H O2 Fe(II) Fe(II) reduced reduced O2 e- come from NADPH via Ferredoxin Reductase/Ferredoxin (Class I enzymes) or NADPH via Cytochrome P450 oxidoreductase (Class II enzymes).  The ordered reaction mechanism minimises the opportunity for cytochromes P450 to reduce O2 prior to binding the organic substrate. This minimises the production of reactive oxygen species, shown in the blue below, that are harmful to cells. Fe(II) O2 Heme O2- O22- DNA damage Superoxide Peroxide e - Fe(III) protein damage Heme OH - OH. Hydroxyl Hydroxyl apoptosis ion radical In Cytochromes P450 when O2 binds the reduced heme the organic substrate is present and this determines the reaction products. The Catalytic Mechanism Common to Most Cytochromes P450 R3C-OH R3C-H Fe(III) oxidised R3C-H R3C-H O Fe(III) Fe(II) oxidised reduced What is the evidence that O2 can bind to reduced H2O cytochromes P450? e- e- , H + R3C-H R3C-H O2 Fe(II) Fe(II) reduced reduced O2 e- come from NADPH via Ferredoxin Reductase/Ferredoxin (Class I enzymes) or NADPH Cytochrome P450 oxidoreductase (Class II enzymes). Designing an experimental strategy to provide evidence for O2 binding to reduced cytochrome P450. How would you do this?  The Catalytic Mechanism Common to Most Cytochromes P450 R3C-OH R3C-H Fe(III) oxidised R3C-H R3C-H O Fe(III) Fe(II) oxidised reduced H2O e- e- , H + R3C-H R3C-H O2 (CO) Fe(II) Fe(II) reduced reduced O2 (CO) e- come from NADPH via Ferredoxin Reductase/Ferredoxin (Class I enzymes) or NADPH Cytochrome P450 oxidoreductase (Class II enzymes). Learning Outcomes After attending the lectures and pursuing private study that involves reading of relevant text-books, on-line resources and peer-reviewed literature, you will be able to: a) demonstrate understanding of the role of UV-vis spectroscopy in: - identifying the heme cofactor in cytochromes P450 - quantifying the redox activity of cytochromes P450 - demonstrating that a diatomic gas can bind to reduced cytochromes P450 b) describe the mechanism common to most cytochromes P450 and illustrate how information from UV-vis spectroscopy lends support to the sequence of events in the catalytic cycle. References can be found in the slides for the first lecture.