Oxygen activation for biological chemistry: The role of redox enzymes

1. Oxygen activation for biological chemistry:The role of redox enzymes Pochapsky Lab Brandeis University

2. Free energy available to prevent equilibration with oxidizing environment h “Fruits” of photosynthesis are reducing equivalents. RIP O2

3. The only thing standing between YOUand Spontaneous human combustion is triplet oxygen

4. Oxygen activation requires spin pairing (metal ion or organic radical) • Cytochrome P450 monooxygenases • Camphor hydroxylase (CYP101) • Drug metabolism (CYP2B4) • Acireductone dioxygenases

5. + 2 H + O 2 N A D H r Pdx - 2 e o Pdx + + NAD + H O H H O 2 Camphor hydroxylase system (CYP101, cytochrome P450cam) O Pdr CYP101 O

6. Cytochrome P450cam- putidaredoxin (Pdx) effector-enzyme complex No change Perturbed Gone Pochapsky et al., Biochemistry 42, 6549 (2003)

7. Ahhh. That’s better. A high-barrier conformational shift occurs upon binding of effector Pdx to P450, switching substrate orientation to the right one for chemistry. Wrong conformation! Wei, Pochapsky & Pochapsky J. Am. Chem. Soc.127, 6974 (2005)

8. Pdx binding site B’ helix 88-89 Ile-Pro Is Pro cis/trans isomerization the switch for conformational change? Crystallography only shows what crystallizes!

9. Tools that we use…. • Site-directed mutagenesis. • Enzymology (turnover and kinetics) • Organic synthesis (isotopically labeled compounds) • NMR and spectroscopy. • Mass spectrometry (local dynamics)

10. Enzymes of the methionine salvage pathway • Many cancer cell lines require methionine supplements, unlike their parent cell lines. • Labeling studies link requirement to the methionine salvage pathway (MSP), that returns the g-thiomethyl of adenosylmethionine to methionine.

11. Methionine salvage pathway

12. One protein, two enzymes:acireductone dioxygenase (ARD) Ni-ARD Fe-ARD Pochapsky et al.,Nature Struct. Biol.9, 966 (2002) Pochapsky et al., J. Biomol. NMR 34, 117-127 (2006) Ju et al., J. Mol. Biol. In press.

13. Synthesis of ARD substrate analogues

14. Human ARD (ALP) • Two fractions are obtained, each showing different types of ARD activity in vitro (catalyze acireductone oxidation). Activity assay for acireductone dioxygenase activity by ALP. Solid points, aerobic treatment of acireductone (lmax=305 nm) with cell-free extract from E. coli overexpressing ALP. Control assay (no overexpression) are shown as empty points.

15. Is ARD the leak in the methionine salvage pathway in cancer cells? • ARD upregulated in some human cancer cell lines, downregulated in others. • ARD binds to matrix metalloproteinase, regulating activity. • CO has messenger activity similar to NO • CO may be anti-apoptotic

16. ????

17. NCO optimized for detection of paramagnetically relaxed resonances in Ni-ARD Kostic et al., J. Am. Chem. Soc. 124, 9054-9055 (2002).

18. 600 MHz INOVA (Edison NMR Lab, Basement of Edison-Lecks) 500 MHz INOVA (Kalman Bldg.) BrUNMR 400 MHz NMR (Friedland Bldg.)

19. Now up and running….. BrUNMR introduces the 800 MHz NMR!