Spectroscopic and computational characterization of substrate-bound mouse cysteine dioxygenase: nature of the ferrous and ferric cysteine adducts and mechanistic implications

Abstract

Cysteine dioxygenase (CDO) is a mononuclear non-heme Fe-dependent dioxygenase that catalyzes the initial step of oxidative cysteine catabolism. Its active site consists of an Fe(II) ion ligated by three histidine residues from the protein, an interesting variation on the more common 2-His-1-carboxylate motif found in many other non-heme Fe(II)-dependent enzymes. Multiple structural and kinetic studies of CDO have been carried out recently, resulting in a variety of proposed catalytic mechanisms; however, many open questions remain regarding the structure/function relationships of this vital enzyme. In this study, resting and substrate-bound forms of CDO in the Fe(II) and Fe(III) states, both of which are proposed to have important roles in this enzyme's catalytic mechanism, were characterized by utilizing various spectroscopic methods. The nature of the substrate/active site interactions was also explored using the cysteine analogue selenocysteine (Sec). Our electronic absorption, magnetic circular dichroism, and resonance Raman data exhibit features characteristic of direct S (or Se) ligation to both the high-spin Fe(II) and Fe(III) active site ions. The resulting Cys- (or Sec-) bound species were modeled and further characterized using density functional theory computations to generate experimentally validated geometric and electronic structure descriptions. Collectively, our results yield a more complete description of several catalytically relevant species and provide support for a reaction mechanism similar to that established for many structurally related 2-His-1-carboxylate Fe(II)-dependent dioxygenases.

Figure 1

RT Abs (top) and 4.5 K MCD (bottom) spectra of as-isolated CDO (– - –), Fe(II)-CDO + Cys (- - -), and as-isolated CDO + Cys (—). Inset: VTVH MCD data (—) collected for Fe(II)-CDO + Cys at 30,580 cm⁻¹ (position indicated by arrow) and 2.2, 4, 8, 15, 25 K. Open circles represent a theoretical fit of the data obtained with the parameters g = 2, S = 2, D = −13.0 cm⁻¹, E/D = 0.23.

Figure 2

77 K rR spectrum of as-isolated CDO + Cys, obtained with λ_ex = 647.1 nm. Features marked by * are due to vibrations of the ice lattice.

Figure 3

RT Abs (top) and 4.5 K MCD (bottom) spectra of as-isolated CDO + Cys (- - -) or Sec (—). Horizontal arrows depict the marked red-shifts of features upon substitution of Cys with Sec. Inset: VTVH MCD data (—) collected for Fe(II)-CDO + Sec at 28,818 cm⁻¹ (position indicated by vertical arrow) and 2, 4, 8, 15, 25 K. Open circles represent a theoretical fit of the data obtained with the parameters g = 2, S = 2, D = 9.0 cm⁻¹, E/D = 0.25.

Figure 4

77 K rR spectrum of as-isolated CDO + Sec, obtained with λ_ex = 752.5 nm. Features marked by * are due to vibrations of the ice lattice.

Figure 5

DFT-optimized active-site model of Cys- (or Sec-) bound Fe(III)-CDO. H atoms have been omitted for clarity.

Figure 6

Relevant portions of the DFT-computed MO diagrams for Cys- and Sec-bound Fe(III)-CDO. Only the spin-down MOs are shown. The S(Se) → Fe CT transitions are indicated by arrows.

Figure 7

Relevant portions of the DFT-computed MO diagrams for Cys- and Sec-bound Fe(II)-CDO. Only the spin-down MOs are shown. The S(Se) → Fe CT transitions are indicated by arrows.

Figure 8

4.5 K MCD spectra of as-isolated (left) and reduced (right) CDO + Cys before (—) and after (---) exposure to O₂.