Characterization of the Nitrosyl Adduct of Substrate-Bound Mouse Cysteine Dioxygenase by Electron Paramagnetic Resonance:  Electronic Structure of the Active Site and Mechanistic Implications

Abstract

Mammalian cysteine dioxygenase (CDO) is a non-heme iron metalloenzyme that catalyzes the first committed step in oxidative cysteine catabolism. The active site coordination of CDO comprises a mononuclear iron ligated by the Nepsilon atoms of three protein-derived histidines, thus representing a new variant on the 2-histidine-1-carboxylate (2H1C) facial triad motif. Nitric oxide was used as a spectroscopic probe in investigating the order of substrate-O2 binding by EPR spectroscopy. In these experiments, CDO exhibits an ordered binding of l-cysteine prior to NO (and presumably O2) similar to that observed for the 2H1C class of non-heme iron enzymes. Moreover, the CDO active site is essentially unreactive toward NO in the absence of substrate, suggesting an obligate ordered binding of l-cysteine prior to NO. Typically, addition of NO to a mononuclear non-heme iron center results in the formation of an {FeNO}7 (S = 3/2) species characterized by an axial EPR spectrum with gx, gy, and gz values of approximately 4, approximately 4, and approximately 2, respectively. However, upon addition of NO to CDO in the presence of substrate l-cysteine, a low-spin {FeNO}7 (S = 1/2) signal that accounts for approximately 85% of the iron within the enzyme develops. Similar {FeNO}7 (S = 1/2) EPR signals have been observed for a variety of octahedral mononuclear iron-nitrosyl synthetic complexes; however, this type of iron-nitrosyl species is not commonly observed for non-heme iron enzymes. Substitution of l-cysteine with isosteric substrate analogues cysteamine, 3-mercaptopropionic acid, and propane thiol did not produce any analogous {FeNO}7 signals (S = 1/2 or 3/2), thus reflecting the high substrate specificity of the enzyme observed by a number of researchers. The unusual {FeNO}7 (S = 1/2) electronic configuration adopted by the substrate-bound iron-nitrosyl CDO (termed {ES-NO}7) is a result of the bidentate thiol/amine coordination of l-cysteine in the NO-bound CDO active site. DFT computations were performed to further characterize this species. The DFT-predicted geometric parameters for {ES-NO}7 are in good agreement with the crystallographically determined substrate-bound active site configuration of CDO and are consistent with known iron-nitrosyl model complexes. Moreover, the computed EPR parameters (g and A values) are in excellent agreement with experimental results for this CDO species and those obtained from comparable synthetic {FeNO}7 (S = 1/2) iron-nitrosyl complexes.

Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of Activity

“…Pierce et al (29) recently reported that, in the absence of cysteine, the iron metallocenter of CDO is essentially unreactive to NO (used as a surrogate probe for O 2 ). Binding of NO, however, is greatly facilitated when CDO is incubated in the presence of cysteine.…”

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

“…An iron content of 0.42 Ϯ 0.028 mol Fe(II) per mol CdoB was determined by the method of Pierce et al (9). Also, Fe(III) could be detected in the purified His 6 -tagged CdoB at 0.048 Ϯ 0.024 mol Fe(III) per mol enzyme.…”

“…Determination of the iron content was done with bathophenanthroline disulfonic acid disodium salt (BP-DADS), based on the method of Pierce et al (9).…”

“…An iron content of 0.38 Ϯ 0.04 mol Fe(II) per mol CdoA was revealed by the method of Pierce et al (9). No Fe(III) could be detected in CdoA.…”

“…Whereas most of the nonheme iron proteins coordinate the metal via two histidine residues and a carboxylic acid group (the 2-His-1-carboxylate facial triad), the ferrous iron in Cdos is arranged in a mutually cis geometry consisting of three histidine residues (3-His facial triad) (1,8,9). The loss of Cdo activity after immobilized metal chelate affinity chromatography (IMAC) purification was reported in several studies (10)(11)(12), and the activity could be reconstituted only by addition of exogenous ferrous iron, whereas other transition metals failed to restore the activity.…”