Chapter 5 References

1. Skaar, E. P.; Gaspar, A. H.; Schneewind, O. (2004) IsdG and IsdI, heme- degrading enzymes in the cytoplasm of Staphylococcus aureus. J. Biol. Chem. 279, 436-443.

2. Conger, M. A.; Pokhrel, D.; Liptak, M. D. (2017) Tight binding of heme to Staphylococcus aureus IsdG and IsdI precludes design of a competitive inhibitor. Metallomics. 9, 556-563.

3. Loutet, S. A.; Kobylarz, M. J.; Chau, C. H.; Murphy, M. E. P. (2013) IruO is a reductase for heme degradation by IsdI and IsdG proteins in Staphylococcus aureus. J. Biol. Chem. 288, 25749-25759.

4. Takayama, S. J.; Loutet, S. A.; Mauk, A. G.; Murphy, M. E. (2015) A Ferric- Peroxo Intermediate in the Oxidation of Heme by IsdI. Biochemistry. 54, 2613- 2621.

5. Streit, B. R.; Kant, R.; Tokmina-Lukaszewska, M.; Celis, A. I.; Machovina, M. M.; Skaar, E. P.; Bothner, B.; DuBois, J. L. (2016) Time-resolved Studies of IsdG Protein Identify Molecular Signposts along the Non-canonical Heme Oxygenase Pathway. J. Biol. Chem. 291, 862-871.

6. Reniere, M. L.; Ukpabi, G. N.; Harry, S. R.; Stec, D. F.; Krull, R.; Wright, D. W.; Bachmann, B. O.; Murphy, M. E. P.; Skaar, E. P. (2010) The IsdG-family of haem oxygenases degrades haem to a novel chromophore. Mol. Microbiol. 75, 1529-1538.

7. Wu, R.; Skaar, E. P.; Zhang, R.; Joachimiak, G.; Gornicki, P.; Schneewind, O.; Joachimiak, A. (2005) Staphylococcus aureus IsdG and IsdI, heme-degrading enzymes with structural similarity to monooxygenases. J. Biol. Chem. 280, 2840- 2846.

8. Takayama, S. J.; Ukpabi, G.; Murphy, M. E.; Mauk, A. G. (2011) Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI. Proc. Natl. Acad. Sci. 108, 13071-13076.

9. Ukpabi, G.; Takayama, S. J.; Mauk, A. G.; Murphy, M. E. P. (2012) Inactivation of the heme degrading enzyme IsdI by an active site substitution that diminishes heme ruffling. J. Biol. Chem. 287, 34179-34188.

10. Lockhart, C. L.; Conger, M. A.; Pittman, D. S.; Liptak, M. D. (2015) Hydrogen bond donation to the heme distal ligand of Staphylococcus aureus IsdG tunes the electronic structure. J. Biol. Inorg. Chem. 20, 757-770.

11. Matsui, T.; Kim, S. H.; Jin, H.; Hoffman, B. M.; Ikeda-Saito, M. (2006)

Compound I of Heme Oxygenase Cannot Hydroxylate its Heme meso-Carbon. J. Am. Chem. Soc. 128, 1090-1091.

12. Poulos, T. L. (2014) Heme enzyme structure and function. Chem. Rev. 114, 3919- 3962.

13. Moody, P. C. E.; Raven, E. L. (2018) The Nature and Reactivity of Ferryl Heme in Compounds I and II. Acc. Chem. Res. 51, 427-435.

14. Rittle, J.; Green, M. T. (2010) Cytochrome P450 Compound I: Capture, Characterization, and C–H Bond Activation Kinetics. Science 330, 933-937. 15. Dunford, H. B.: Peroxidases and Catalases: Biochemistry, Biophysics,

Biotechnology, and Physiology; 2nd ed.; John Wiley and Sons: New York, 2010. 16. Yosca, T. H.; Rittle, J.; Krest, C. M.; Onderko, E. L.; Silakov, A.; Calixto, J. C.;

Behan, R. K.; Green, M. T. (2013) Iron(IV)hydroxide pKa and the Role of

Thiolate Ligation in C-H Bond Activation by Cytochrome P450. Science 342, 825-829.

17. Yosca, T. H.; Langston, M. C.; Krest, C. M.; Onderko, E. L.; Grove, T. L.; Livada, J.; Green, M. T. (2016) Spectroscopic Investigations of Catalase

Compound II: Characterization of an Iron(IV) Hydroxide Intermediate in a Non- thiolate-Ligated Heme Enzyme. J. Am. Chem. Soc. 138, 16016-16023.

18. Yosca, T. H.; Behan, R. K.; Krest, C. M.; Onderko, E. L.; Langston, M. C.; Green, M. T. (2014) Setting an upper limit on the myoglobin iron(IV)hydroxide pK(a): insight into axial ligand tuning in heme protein catalysis. J. Am. Chem. Soc. 136, 9124-9131.

19. Zeng, W.; Barabanschikov, A.; Zhang, Y.; Zhao, J.; Sturhan, W.; Alp, E. E.; Sage, J. T. (2008) Synchotron-Derived Vibrational Data Confirm Unprotonated Oxo Ligand in Myoglobin Compound II. J. Am. Chem. Soc. 130, 1816-1817. 20. Kwon, H.; Basran, J.; Casadei, C. M.; Fielding, A. J.; Schrader, T. E.; Ostermann,

A.; Devos, J. M.; Aller, P.; Blakeley, M. P.; Moody, P. C.; Raven, E. L. (2016) Direct visualization of a Fe(IV)-OH intermediate in a heme enzyme. Nat. Commun. 7, 13445.

21. Kapust, R. B.; Tozser, J.; Fox, J. D.; Anderson, D. E.; Cherry, S.; Copeland, T. D.; Waugh, D. W. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 14, 993-1000.

22. Graves, A. B.; Horak, E. H.; Liptak, M. D. (2016) Dynamic ruffling distortion of the heme substrate in non-canonical heme oxygenase enzymes. Dalton Trans. 45, 10058-10067.

23. Nallamsetty, S.; Kapust, R. B.; Tözsér, J.; Cherry, S.; Tropea, J. E.; Copeland, T. D.; Waugh, D. S. (2004) Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro. Protein Expr. Purif. 37, 108-115.

24. Browett, W. R.; Fucaloro, A. F.; Morgan, T. V.; Stephens, P. J. (1983) Magnetic Circular Dichroism Determination of Zero-Field Splitting in Chloro(meso- tetraphenylporphinato)iron(III). J. Am. Chem. Soc. 105, 1868-1872.

25. Browett, W. R.; Gasyna, Z.; Stillman, M. J. (1988) Temperature Dependence and Electronic Transition Energies in The Magnetic Circular Dichroism Spectrum of Horseradish Peroxidase Compound I. J. Am. Chem. Soc. 110, 3633-3640.

26. Lee, W. C.; Reniere, M. L.; Skaar, E. P.; Murphy, M. E. (2008) Ruffling of metalloporphyrins bound to IsdG and IsdI, two heme-degrading enzymes in Staphylococcus aureus. J. Biol. Chem. 283, 30957-30963.

27. Guex, N.; Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer: An Envrionment for Comparative Protein Modeling. Electrophoresis 18, 2714-2723. 28. Hanwell, M. D.; Curtis, D. E.; Lonie, D. C.; Vandermeersch, T.; Zurek, E.;

Hutchison, G. R. (2012) Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J. Cheminform. 4, 17.

29. Fonseca Guerra, C.; Snijders, J. G.; te Velde, G.; Baerends, E. J. (1998) Towards an Order-N DFT Method. Theor. Chem. Acc. 99, 391-403.

30. te Velde, G.; Bickelhaupt, E. M.; Baerends, E. J.; Guerra, C. F.; van Gisbergen, S. J. A.; Snijders, J. G.; Ziegler, T. (2001) Chemistry with ADF. J Comput Chem 22, 931-967.

31. ADF2017. SCM, Theoretical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands, http://www.scm.com/.

32. Swart, M. (2003) AddRemove: A New Link Model for Use in QM/MM Studies. Int. J. Quantum Chem. 92, 177-183.

33. Swart, M.; van Duijnen, P. T.; Snijders, J. G. (2001) A Charge Analysis Derived from an Atomic Multipole Expansion. J. Comput. Chem. 22, 79-88.

34. Perdew, J. P.; Burke, K.; Ernzerhof, M. (1996) Generalized Gradient Approximation Made Simple. Phys. Rev. Let. 77, 3865-3868.

35. Van Lenthe, E.; Baerends, E. J. (2003) Optimized Slater-Type Basis Sets for the Elements 1-118. J. Comput. Chem. 24, 1142-1156.

36. Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz Jr., K. M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. (1995) A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules. J. Am. Chem. Soc. 117, 5179-5197.

37. Jentzen, W.; Song, X. Z.; Shelnutt, J. A. (1997) Structural Characterization of Synthetic and Protein-bound Porphyrins in Terms of the Lowest-Frequency Normal Coordinates of the Macrocycle. J. Phys. Chem. B 101, 1684-1699. 38. Graves, A. B.; Graves, M. T.; Liptak, M. D. (2016) Measurement of Heme

Ruffling Changes in MhuD Using UV-vis Spectroscopy. J. Phys. Chem. B 120, 3844-3853.

39. Neese, F. (2012) The ORCA program system. Wiley Interdiscip. Rev. Comput. Mol. Sci. 2, 73-78.

40. Weigend, F.; Ahlrichs, R. (2005) Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy. Phys. Chem. Chem. Phys. 7, 3297-3305.

41. Vickery, L.; Nozawa, T.; Sauer, K. (1976) Magnetic Circular Dichroism Studies of Myoglobin Complexes. Correlations with Heme Spin State and Axial Ligation. J. Am. Chem. Soc. 98, 343-350.

42. Cheesman, M. R.; Greenwood, C.; Thomson, A. J. (1991) Magnetic Circular Dichroism of Hemoproteins. Adv. Inorg. Chem. 36, 201-255.

43. Bolington, D. G.; Gadsby, P. M. A.; Sievers, G.; Peterson, J.; Thompson, A. J. (1983) A Comparative Study of the Low-Temperature Magnetic Circular Dichroism Spectra of Horse Heart Metmyoglobin and Bovin Liver Catalase Derivatives. Biochim. Biophys. Acta 742, 648-658.

44. Graves, A. B.; Morse, R. P.; Chao, A.; Iniguez, A.; Goulding, C. W.; Liptak, M. D. (2014) Crystallographic and spectroscopic insights into heme degradation by Mycobacterium tuberculosis MhuD. Inorg. Chem. 53, 5931-5940.

45. Browett, W. R.; Stillman, M. J. (1980) Magnetic Circular Dichroism Studies on the Electronic Configuration of Catalase Compounds I and II. Biochim. Biophys. Acta 623, 21-31.

46. Foote, N.; Gadsby, P. M. A.; Field, R. A.; Greenwood, C.; Thomson, A. J. (1987) A Comparison by Magnetic Circular Dichroism of Compound X and Compound II of Horseradish Peroxidase. FEBS Lett. 214, 347-350.

47. Foote, N.; Gadsby, P. M. A.; Greenwood, C.; Thomson, A. J. (1989) pH- dependent Forms of the Ferryl Haem in Myoglobin Peroxide Analyzed by Variable-Temperature Magnetic Circular Dichroism. Biochem. J. 261, 515-522. 48. Hersleth, H. P.; Ryde, U.; Rydberg, P.; Gorbitz, C. H.; Andersson, K. K. (2006)

Structures of the high-valent metal-ion haem-oxygen intermediates in peroxidases, oxygenases and catalases. J. Inorg. Biochem. 100, 460-476.

49. Behan, R. K.; Green, M. T. (2006) On the status of ferryl protonation. J. Inorg. Biochem. 100, 448-459.

50. Wang, X.; Ullrich, R.; Hofrichter, M.; Groves, J. T. (2015) Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive. Proc. Natl. Acad. Sci. 112, 3686- 3691.

51. Pauling, L. (1948) Nature of Forces Between Large Molecules of Biological Interest. Nature 161

52. Hammond, G. S. (1955) A Correlation of Reaction Rates. J. Am. Chem. Soc. 77, 334-338.

53. Richard, J. P. (2013) Enzymatic rate enhancements: a review and perspective. Biochemistry. 52, 2009-2011.

54. Walker, F. A.; Nasri, H.; Turowska-Tyrk, I.; Mohanrao, K.; Watson, C. T.; Shokhirev, N. V.; Debrunner, P. G.; Scheidt, W. R. (1996) π-Acid Ligands in Iron(III) Porphyrinates. Characterization of Low-spin Bis(tert-

butylisocyanide)(porphyrinato)iron(III) Complexes Having (dxz,dyz)4(dxy)1 Ground

State. J. Am. Chem. Soc. 118, 12109-12118.

55. Sitter, A. J.; Reczek, C. M.; Terner, J. (1985) Heme-linked Ionization of Horseradish Peroxidase Compound II Monitored by the Resonance Raman Fe(IV)=O Stretching Vibration. J. Biol. Chem. 260, 7515-7522.

6.1 CONCLUSIONS

The spectroscopic characterization of IsdG presented here has provided insight into heme binding and degradation by IsdG. Equilibrium data confirmed IsdG and IsdI bind heme with nanomolar affinity, placing them well within range to effectively bind heme in the cytosol. The Kd of heme from IsdG is an order of magnitude lower than that from IsdI

based upon the equilibrium data, which was corroborated by competition assays with myoglobin. This data in addition to the fact that the half-life of IsdG, but not IsdI, is increased by the presence of heme in vivo1 led us to propose that IsdG is involved in iron acquisition while IsdI is involved in heme homeostasis, prompting further characterization of IsdG.

A combined Abs, CD, and MCD study of ferric-cyanoheme and ferric-azidoheme derivatives of IsdG was carried out to elucidate the interaction of Asn7 with the distal ligand. Introduction of the N7A mutation altered the electronic structure of ferric- azidoheme but not ferric-cyanoheme, confirming a hydrogen-bond exists between Asn7 and the iron-ligating (α) atom of the distal ligand. We set to quantify the strength of the Asn7…_N

3 hydrogen-bond with equilibrium measurements, and found loss of Asn7 changed

the free energy by ~1 kcal/mol. Given the significant electrostatic component of a hydrogen-bond, an NH…O hydrogen-bond in ferric-peroxoheme is expected to be significantly stronger than an NH…N hydrogen-bond in ferric-azidoheme. DFT predicted that this hydrogen-bond triggers rotation of the distal ligand, which was corroborated by CD. Finally, DFT predicted Ans7-induced spin density delocalization from Fe onto the heme meso carbons, but there was no experimental evidence to support this proposal.

EPR and NMR were used to provide insight into the electronic and spin distribution changes to IsdG–heme–N3 triggered by the Asn7…N3 hydrogen-bond. Both MCD and EPR

are consistent with a (dxy)2(dxz,dyz)3 3d ground electron configuration for Fe, but Asn7

mixes 3dxy character into the singly-occupied molecular orbital of WT IsdG–heme–N3.

Mixing of Fe 3dxy and porphyrin a2u is a mechanism known to delocalize spin from Fe onto

the heme meso carbons, which prompted additional studies. NMR is an ideal tool to probe spin density changes, as the chemical shifts of low-spin ferric heme are governed by its spin distribution.2 We found introduction of the N7A mutation shifted the meso carbons upfield ~10 ppm, consistent with a significant reduction in spin density at those nuclei, confirming the DFT-predicted, Asn7-induced spin density delocalization. A QM/MM model confirmed the N7A mutant retains the ruffled heme deformation, suggesting heme ruffling is not the origin of the Asn7-induced spin density delocalization. Instead, The Asn7…N3 hydrogen bond stabilizes Fe 3dxz- and 3dyz-orbitals which increases the Fe 3dxy

and porphyrin a2u character in the singly-occupied molecular orbital (SOMO), allowing

spin delocalization onto the meso carbons. These results suggest IsdG funnels the reactivity of ferric-peroxoheme toward heme hydroxylation through an Asn7-dependent bridged transition state, circumventing production of reactive, uncontrolled intermediates.

Abs and MCD were used to study a ferryl IsdG structurally related to the reactive intermediate formed upon heme hydroxylation through a bridged transition state. Heme hydroxylation inevitably proceeds through a high-valent ferryl intermediate upon ferric- peroxoheme O–O bond homolysis. Optical spectroscopy confirmed the active site of WT IsdG stabilizes a ferryl intermediate. In contrast, the active site of N7A IsdG cannot accommodate a ferryl heme, or destabilizes a ferryl heme beyond detection. Stabilization

of an intermediate ferryl heme by Asn7 lowers the activation barrier of heme hydroxylation. Based upon the data presented here, we propose Asn7 organizes a structure that is conducive to heme hydroxylation by hydrogen-bonding to the distal ligand and triggering spin delocalization onto the meso carbons, then drives the reactive forward by stabilizing a reactive intermediate structurally related to the bridged transition state.