Combined spectroscopic and structural approaches to explore the mechanism of histidine-ligated heme-dependent aromatic oxygenases

Abstract

The emergence of histidine-ligated heme-dependent aromatic oxygenases (HDAOs) has greatly enriched heme chemistry, and more studies are required to appreciate the diversity found in His-ligated heme proteins. This chapter describes recent methods in probing the HDAO mechanisms in detail, along with the discussion on how they can benefit structure-function studies of other heme systems. The experimental details are centered on studies of TyrHs, followed by explanation of how the results obtained would advance the understanding of the specific enzyme and also HDAOs. Spectroscopic methods, namely, electronic absorption and EPR spectroscopies, and X-ray crystallography are valuable techniques commonly used to characterize the properties of the heme center and the nature of heme-based intermediate. Herein, we show that the combination of these tools are extremely powerful, not only because one can acquire electronic, magnetic, and conformational information from different phases, but also because of the advantages brought by spectroscopic characterization on crystal samples.

Keywords: Heme-based intermediate; Histidine-ligated heme enzymes; Protein crystallization; Spectroscopy; Substrate analog.

Fig. 1

Reaction schemes of HDAOs. R₁ = H, H, and Cl, and R₂ = H, CH₃, and H in TDO/IDO, MarE, and PrnB, respectively; R₃ = H or halogen in TyrH, and CH₃ in SfmD.

Fig. 2

A phylogenetic tree and representative crystal structures of HDAOs. Protein clades and structures of prokaryotic TDO, eucaryotic TDO, PrnB, IDO, SfmD, TyrH, Unidentified TDO, and MarE are colored in light blue, dark blue, yellow, gray, light green, red, purple, and dark green, respectively. The heme cofactors, axial ligands, and substrates are represented by deep red, white blue, and yellow sticks, respectively. The structures are from PDB with accession codes, 2NW8, 5TIA, 2X68, 6E46, 6VDQ, 7KQR,

Fig. 3

A simplified molecular diagram showing the absorption bands typically observed in HDAOs.

Fig. 4

Simplified molecular diagrams and structures of (A) high-spin ferric, (B) HALS, (C) reduced, (D) nitrosyl forms of heme observed in HDAOs. The figure is modified from Davis, I., Geng, J., & Liu, A. (2022). Chapter eight—Metalloenzymes involved in carotenoid biosynthesis in plants. In: E. T. Wurtzel (Ed.), Methods in enzymology (Vol. 671, pp. 207–222). Academic Press. https://doi.org/10.1016/bs.mie.2022.01.012.

Fig. 5

TyrH crystals observed under microscope.