Characterization of the nonheme iron center of cysteamine dioxygenase and its interaction with substrates

JBC Papers in Press. Published on June 28, 2020 as Manuscript RA120.013915

The latest version is at https://www.jbc.org/cgi/doi/10.1074/jbc.RA120.013915

Monodentate thiolate coordination of ADO substrates

Characterization of the non-heme iron center of cysteamine dioxygenase and

its interaction with substrates

Yifan Wang^¶, Ian Davis^{¶, ‡}, Yan Chan^{‡, ±}, Sunil G. Naik^†, Wendell P. Griffith^¶, and Aimin Liu*^{,¶, ‡}

From ^¶Department of Chemistry, University of Texas, San Antonio, TX 78249, United States

^‡Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States

^±Current address: Debut Biotechnology, Inc, San Diego, CA

^†Deceased

Running title: Monodentate thiolate coordination of ADO substrates

*To whom correspondence should be addressed. Tel.: 210-458-7062; Fax: 210-458-7428; E-mail:

Feradical@utsa.edu

Keywords: thiol regulation; EPR spectroscopy; nitric oxide; oxygen activation; oxygen sensing,

cysteamine dioxygenase (ADO); cysteine dioxygenase (CDO); sulfur metabolism; thiol dioxygenase.

ABSTRACT:

against oxidative stress. The findings of this

work contribute to the understanding of the

substrate interaction in ADO and fill a gap in

our knowledge of the substrate specificity of

thiol dioxygenases.

Cysteamine dioxygenase (ADO) has been

reported to exhibit two distinct biological

functions with a non-heme iron center. It

catalyzes oxidation of both cysteamine in sulfur

metabolism and N-terminal cysteine-containing

proteins or peptides, such as regulator of G

protein signaling 5 (RGS5). It thereby preserves

oxygen homeostasis in a variety of physiological

processes. However, little is known about its

catalytic center and how it interacts with these

Thiol dioxygenases are a group of non-

heme, ferrous enzymes that incorporate two oxygen

atoms from molecular oxygen into the thiol groups

of their corresponding substrates (1). Among this

group of proteins, cysteamine dioxygenase (ADO)

two types of primary substrates in addition to O₂. (2), cysteine dioxygenase (CDO) (3-6), 3-

Here, using EPR, Mössbauer, and UV-Vis

spectroscopies, we explored the binding mode of

cysteamine and RGS5 to human and mouse

ADO proteins in their physiologically relevant

ferrous form. This characterization revealed

that in the presence of nitric oxide as a spin

probe and oxygen surrogate, both the small

molecule and the peptide substrates coordinate

to the iron center with their free thiols in a

monodentate binding mode, in sharp contrast to

binding behaviors observed in other thiol

dioxygenases. We observed a substrate-bound

B-type dinitrosyl iron center complex in ADO,

suggesting the possibility of dioxygen binding to

the iron ion in a side-on mode. Moreover, we

observed a substrate-mediated reduction of the

ferric to the ferrous oxidation state at the iron

center. Subsequent MS analysis indicated

corresponding disulfide formation of the

substrates, suggesting that the presence of the

substrate could reactivate ADO to defend

mercaptopropionate dioxygenase (3MDO) (7-9),

and mercaptosuccinate dioxygenase (MSDO) (10)

are found to catalyze the oxidation of small

molecules as shown in Figure 1A. To date, ADO

and CDO are the only two known thiol

dioxygenases in humans, and they are directly

involved in cysteine metabolism and the

biosynthesis of hypotaurine and taurine (1).

Dysfunction of these thiol dioxygenases is

associated with oxidative stress, autoimmune

deficiency, and neurodegenerative diseases (11-18).

Additionally, some thiol dioxygenases

oxidize N-terminal cysteine to cysteine sulfinic acid

in polypeptides to regulate protein stability (Figure

1B). For example, plant cysteine oxidase (PCO)

promotes the N-terminal thiol dioxygenation of VII

ethylene response factors in normoxia to proceed

arginylation, resulting in proteasomal degradation

(19). Recently, such

a

posttranslational

modification mediated by thiol dioxygenases is

reported in ADO (20). Analogous to PCO in plants,

1

Monodentate thiolate coordination of ADO substrates

ADO has been proposed to function as a conserved

oxygen sensor in animals, regulating the

degradation of N-cysteine proteins, including

regulator of G signaling 5 (RGS5), and thus,

transduces cellular responses to hypoxia. RGS5

belongs to the R4 subfamily of RGS proteins that

are known to negatively regulate the signaling of G-

protein coupled receptors (21). Enriched in

cardiovascular tissues, RGS5 is related to pericyte

maturation, vascular functions, and hypoxia-

induced apoptosis (22-24). As such an essential

modulator, RGS5 is also one of the most effective

substrates of human ADO with kinetic parameters

k_catof 16.9 s^-1and K_mof 71.5 μM (20). In contrast,

CDO does not possess such a dioxygenation

activity on N-cysteine peptides. Overall, ADO is

not only part of the thiol metabolism cycle,

regulating through its dioxygenase activity of

cysteamine, its function also extends to oxygen

homeostasis and signal transduction in mammals.

The biological significance of ADO is increasingly

appreciated, and thus, exploring its catalytic center

and especially how it responds to small molecule

and protein substrates is needed.

carboxylate groups of cysteine. In addition to the

crosslinked Tyr157, the carboxylate group of the

substrate is also recognized by other second sphere

residues such as Tyr58 and Arg60 (Figure 1C).

Such extensive interactions assist the correct

positioning of cysteine in the process of enzyme-

substrate (ES) complex formation.

Although ADO is a sibling enzyme of CDO

catalyzing a similar reaction, the structural features

and substrate binding of ADO are much less

understood due to the absence of a crystal structure.

However, it is important to characterize how the

ADO active site handles both small molecule and

protein or peptide substrates, a feature that is so far

unique among thiol dioxygenases. Our previous

study has identified the presence of a Cys-Tyr

cofactor in ADO through noncanonical amino acid

incorporation via the genetic code expansion

strategy followed by high-resolution mass

spectrometric analysis (27). In contrast with the

cofactor of CDO, which is formed by two residues

distant from each other (63 residues apart) leading

to a global structural change (30), the Cys-Tyr

cofactor in ADO is formed by two adjacent residues,

Cys220 and Tyr222, and the structures of

crosslinked and uncrosslinked proteins have little

global difference that is undetectable by low-

resolution techniques such as SDS-PAGE (27).

Understandably, a predicted structure of ADO

indicates a different location of the Cys-Tyr

cofactor relative to the iron center of CDO (Figure

1C), on the opposite side of the substrate-binding

cavity. Moreover, amino acid sequence alignment

suggests residues responsible for substrate

recognition in CDO are mostly missing in ADO,

A protein-derived Cys-Tyr cofactor is

autocatalytically generated in mammalian ADO

and CDO to amplify their enzymatic activities

when the substrate level rises (25-27). Such a

substrate-initiated

enzyme

posttranslational

modification and activity amplification is likely a

mechanism for maintaining the physiologically

significant, free thiol-containing substances at a

proper cellular level. The active site geometry and

substrate binding mode of CDO in the ternary

complex with a mature Cys-Tyr cofactor is revealed

by the X-ray crystal structure with nitric oxide (^•NO) except for Tyr58 (Figure S1). Therefore, although

as an O₂surrogate (28). The Cys-Tyr cofactor is

formed by Cys93 and Tyr157 through a thioether

bond located approximately 5 Å from the iron

center in CDO (Figure 1C). Its presence increases

the catalytic rate by ten-fold (25). The substrate

cysteine binds to the iron center in a bidentate

fashion with both its amine and thiol groups.

Together with an end-on bound ^•NO, the iron center

forms octahedral coordination. This active site

architecture in the complex resembles the

persulfenate species that was structurally

characterized in CDO, but its catalytic competence

remains to be established (29). The Cys-Tyr

some structural and mechanistic similarity is

generally anticipated between ADO and CDO

because of the catalytic reactions they promote, the

substrate-binding mode of ADO cannot be simply

extrapolated due to intrinsic differences.

Here, we conducted an EPR-centered

spectroscopic study of ADO from both human and

mouse origin. Two proteins share 85.6% amino

acid sequence identity (Figure S1). We have found

that the substrate of the ADO, either cysteamine or

the N-terminus of RGS5, is capable of reducing the

iron center by forming a disulfide product if the

enzyme is in the ferric oxidation state. The

spectroscopic data also reveal a new binding mode

of the substrate, monodentate coordination through

•

cofactor of CDO not only interacts with NO but

also forms hydrogen bonds with both the amine and

2

Monodentate thiolate coordination of ADO substrates

the thiol group. This study fills the gap in the

substrate specificity of the thiol dioxygenases

catalyzing a reaction without a free carboxylate

group.

fraction of g = 4.30 remained, and all other

resonances were no longer detectable. This

experiment confirmed that cysteamine can reduce

the ferric ADO to the EPR silent ferrous state.

Next, the same anaerobic incubation was

Results

done with the peptide substrate. The first 14 amino

acids of the Met-excised N-terminus of RGS5 was

chemically synthesized with a sequence of

CKGLAALPHSCLER following a previous study

(20). A similar phenomenon was observed when the

N-terminus of RGS5 was incubated with ADO

(Figure 2B). After incubation with the peptide

substrate for 1 min, the high-spin ferric signal

centered at g = 4.30 was converted to a new signal

at g = 4.42 and 4.26 with a decreased ferric

population; small resonances at g = 9.13 and 5.02

were present. These new signals have different g

values compared with the samples incubated with

cysteamine, which suggests the electronic structure

of ferric iron center was different upon cysteamine

or peptide ligation, while both substrates could

reduce Fe^III-ADO. At longer incubation time of 3

and 6 min, the primary ferric signal, as well as the

minor resonances, continuously decreased. After a

10-min incubation, only a small fraction of the

major ferric species remained oxidized, and the

minor resonances were nearly absent. Together, the

above results revealed that both the cysteamine and

N-terminal cysteine-containing peptide could

readily reduce the ferric center of ADO through

their corresponding thiol groups, implicating

thiolate ligation in both cases.

Enzyme reactivation through chemical reduction of

the ferric iron center by the primary substrate–

Considering that cysteamine, as well as the

cysteine-containing peptide substrates, contains a

free thiol, we tested if these ADO substrates can

reduce the ferric form of the enzyme to the ferrous

state. X-band continuous-wave EPR spectroscopy

was used to follow the change of the oxidation state

of the iron center upon incubation with cysteamine

or N-terminus of RGS5. After an overnight air

exposure at 4 °C, the as-purified ADO became

oxidized, and the EPR spectrum showed a notable

species with a central g value of 4.30. This g = 4.30

feature originates from the middle doublet of a

rhombic (E/D ~1/3), high-spin (S = 5/2) non-heme

ferric iron coordinated by protein ligands (Figure

2A, black trace). A few minor EPR signals were

observed at g = 9.31, 7.25, and 5.82. The g = 9.31

feature can be attributed to the overlapping

resonances within the lowest and highest lying

doublets of the S = 5/2 spin system, whereas other

minor features are likely due to inhomogeneity of

the iron center that will be discussed later. After

anaerobically mixing with the substrate cysteamine

and incubating for 1 min, the major ferric EPR

signal of the oxidized ADO was diminished, and its

resonance at g = 4.30 broadened slightly (Figure

2A). Additionally, new spectral features at g = 9.22

and 4.73 arose, concomitant with the disappearance

of the minor species observed in the spectrum of

Fe^III-ADO. The noticeable shift of the g value and

the appearance of new resonance after cysteamine

addition are tentatively attributed to substrate

ligation to the ferric iron center. Similar EPR

features have been observed in samples of as-

isolated desulfoferrodoxin (36) and neelaredoxin

(37), two non-heme iron-binding proteins

coordinated by cysteinyl-sulfur ligands. Hence,

based on literature precedence, the thiol moiety of

cysteamine is expected to participate in iron

coordination, although at this stage, it is not

definitive. After 3- and 6-min substrate incubation,

the high-spin ferric signal at g = 4.30 continuously

decreased, along with the resonances at g = 9.22

and 4.73. After a 10-min incubation, only a minor

It was previously unknown that the primary

substrate of ADO can reduce its ferric ion to the

catalytically active ferrous form. To verify this

finding, especially the disappearance of the non-

heme ferric ion EPR signal upon substrate binding

was due to the change of the metal oxidation state

rather than the relaxation caused spectral line

broadening of the EPR signal, a separate set of EPR

experiments was conducted on mouse ADO

(Figure 3A). The mouse ADO lacks 12 amino acids

that present in the N-terminus of the human protein.

However, this region is not directly related to the

active site (Figure S1). The mouse protein can

tolerate chemical oxidation. Thus, ferricyanide was

employed to oxidize the as-isolated ADO for 5 h.

After removing excessive oxidant by desalting and

buffer change, the oxidized ADO (Figure 3B) was

made anaerobic and mixed with 50 mM cysteamine

under O₂-free conditions. A putative cysteamine-

3

Monodentate thiolate coordination of ADO substrates

bound high-spin ferric species was shown

concomitantly with a reduction of the EPR signal

(Figure 3 C-D). Finally, the high-spin ferric EPR

signal of the oxidized ADO decreased slowly and

completely disappeared after 20 min of cysteamine

incubation (Figure 3E). Exposure of the substrate-

reduced ADO to nitric oxide (^•NO) under O₂-free

conditions led to the formation of an Fe(II)-nitrosyl

complex (Figure 3F), whose electronic structure

will be further characterized below. Together, the

results from human ADO and mouse ADO

unambiguously demonstrate that the substrate of

the enzyme is capable of reducing the catalytically

inactive ferric center to the enzymatically active

ferrous state.

indicates that there was still one ferric and two

ferrous species present in ADO upon substrate

incubation. The quadrupole splitting and isomer

shift of the ferric species (red trace) had no

measurable change; however, its population

decreased to 14%. The Fe^II_Aspecies (purple trace)

also maintained the same δ and ΔE_Qvalues but with

a reduced population. Notably, the Fe^II_Bspecies in

the resting ADO (green trace) completely

disappeared, concomitant with the appearance of a

new, predominant ferrous species termed Fe^II

C

(blue trace, δ_C= 1.19 mm/s, ΔE_QC= 2.85 mm/s) that

comprises 72% of the iron content. The Mössbauer

parameters are summarized in Table 1. This

observation suggests that Fe^IIis directly

B

responsible for the substrate binding and reduction,

and the newly evolved Fe^IIis the binary ES

Characterization of the ADO iron center by

Mössbauer spectroscopy–To elaborate the

substrate-induced iron reduction and analyze the

iron inhomogeneity in the ligand-free ADO by an

independent technique, we executed a Mössbauer

spectroscopic characterization of the iron center of

as-isolated mouse ADO. The concentrated, as-

isolated ADO protein was analyzed by Mössbauer

spectroscopy at 4.2 K. For resting ADO, the

observed zero-field Mössbauer spectrum was fitted

to three species. As shown in Figure 4A, ~23% of

the total iron was assigned to a ferric doublet (red

trace, δ = 0.27 mm/s; ΔE_Q= 0.47 mm/s) and the

remaining 77% was in the ferrous state. The 77%

ferrous iron was composed of two quadrupole

C

complex. Moreover, the addition of cysteamine

induces homogeneity of the iron species by

converting the inactive species, Fe^IIIand Fe^II, to

A

the active form Fe^II.

B

By subtracting the fitted signals of Fe^IIIand

Fe^IIfrom the experimental spectra, the spectra of

A

active enzyme alone (green line) and ES complex

(blue line) are shown in Figure 4C. Comparing the

subtracted spectra reveals a change to both isomer

shift and quadrupole splitting of the iron center

upon substrate binding. The decrease of the δ value,

from 1.26 mm/s of the substrate-free enzyme to

1.19 mm/s of the ES complex, agrees with sulfur

atom coordination to the ferrous center (9,38,39).

doublets, Fe^II(purple trace, δ_A= 1.16 mm/s,

A

ΔE_Q(A)= 2.45 mm/s) and Fe^II_B(green trace, δ_B= 1.26

mm/s, ΔE_Q(B)= 2.93 mm/s) with the ratio of 1 : 2.2.

The quadrupole doublets and isomer shift values of

Fe^IIand Fe^IIsuggest a high-spin ferrous iron

Detection of by-products in the substrate-mediated

enzyme reactivation

Based on the Mössbauer and EPR analyses, the

reduction of Fe^III-ADO occurred through the

interaction between the thiol of the substrate and the

iron center. The reactivation of ADO from the ferric

form to the catalytically active ferrous form is at the

expense of substrate oxidation. The process

forming disulfide product could proceed through

the following reaction (Eq. 1):

A

B

coordinated by N- and O-atom ligands, forming

penta- or hexa-coordination. All thiol dioxygenases

known so far possess a 3-His motif as the first

sphere metal-coordination in a cupin fold (1), and

this is also expected to be the case for ADO (27).

Hence, the iron center presumably contains two or

three solvent-derived ligands. Also, the multiple

species observed suggest that the geometry of the

iron center is inhomogeneous. The Mössbauer

spectrum is largely in accord with the results

reported for other thiol dioxygenases with 3-His

coordination motif (9,31,38).

Fe^III-ADO + 2 R–SH ⟶ Fe^II-ADO + R–S–S–R

(1)

To investigate whether a fraction of the substrate

forms R-S-S-R, cysteamine was anaerobically

incubated with the oxidized mouse ADO at room

temperature for one hour. Cysteamine is known to

be readily oxidized to cystamine, which is a

dimerized product with a disulfide bridge. Thus,

commercially purchased cysteamine unavoidably

To pinpoint how the iron center responds to

the addition of cysteamine, the binary complex was

characterized (Figure 4B). The spectral fitting

4

Monodentate thiolate coordination of ADO substrates

contains cystamine impurity. A control sample was

prepared with cysteamine in the absence of ADO.

cysteines. While the population of the

intramolecular disulfide peptide was overwhelming

Samples were then filtered and analyzed by LC-MS. compared with the intermolecular disulfide peptide.

As shown in Figure S2, although a portion of

cysteamine was already dimerized and eluted as

cystamine in the control, the amount of cystamine

had a noticeable increase after anaerobic incubation

with the oxidized enzyme. This shows that the

oxidized product is indeed the substrate dimer with

a disulfide bond.

It is worth mentioning that a nonspecific dimer of

the intramolecular disulfide peptide was also

observed but with relatively low intensity, and its

m/z peaks were partially overlapped with the ones

of the intermolecular crosslinked dimer. The

presence of both intra- and intermolecular disulfide

peptides suggests that after activation in the active

site of ADO, the thiol group of the peptide substrate

diffuses out to the solution and reacts with another

free thiol to form the disulfide product. It is more

favored when encountering the thiol within the

same peptide chain. Since the RGS5 peptide did not

contain crosslinked dimer in controls, but only after

ADO reactivation, these results provide strong

support for Eq. 1. Notably, other than disulfide

products, the highly reactive substrate thiol radicals

also randomly reacted with surface cysteine

residues to form a disulfide bond, as we observed

by intact protein mass spectrometry. For such a

reason, substrate or product quantitation was not

further pursued to investigate the precise

stoichiometry of the reduction.

The most appealing conclusion was,

however, derived from the experiment with the

peptide substrate. The N-terminal peptide contains

14 amino acids (CKGLAALPHSCLER), which

should have a larger steric hindrance than

cysteamine. Although we did observe the N-

terminal RGS5 peptide could readily reduce the

iron center and reactivate the ferric form of ADO to

the catalytically active form, if the dimeric peptide

can still be formed as the product is in question. We

next executed the experiment with the RGS5

peptide and human ADO. Similarly, RGS5 (5 mM)

was anaerobically incubated with the oxidized

ADO for one hour and analyzed using high-

resolution MS. The control of peptide alone

generated a spectrum of pure RGS5 peptide with

peaks carrying different charges (Figure 5A). The

m/z values of 375.1975, 499.9274, and 749.3871

represents the intact peptide at +4, +3, and +2

charge states, respectively, with a mass error of less

than 1.1 ppm. The heavier species shown at m/z of

Cysteamine alters the binding behavior of nitric

oxide to the non-heme iron center of ADO

In the resting state, non-heme iron-dependent thiol

dioxygenases contain a mononuclear ferrous iron

coordinated by three histidine residues in their

active site. Unlike heme-dependent iron proteins,

which have unique UV-vis, EPR, and other

spectroscopic features, these non-heme iron centers

lack representative spectroscopic characteristics to

monitor the process of substrate binding and

reaction turnovers. Historically, in spectroscopic

999.1814 corresponds to

a dimer of two

nonspecifically interacted peptide molecules at the

+3 charge state. After incubation with the oxidized

enzyme, the mass spectrum of the peptide appeared

differently (Figure 5B). The peaks with m/z of

374.6933, 499.2552, and 748.3788 are from a

modified peptide at +4, +3, and +2 charge states,

respectively (marked in red). The loss of 2 Da

compared with the intact peptide indicates the

modified peptide was two-proton less (mass error

less than 0.3 ppm). Considering there is another

cysteine residue present in the peptide besides the

N-terminal cysteine, most likely, an intramolecular

disulfide bond was formed between these two

cysteines. Additionally, peaks with m/z of 599.3061

and 998.1714 were also found in the spectrum

(marked in blue), corresponds to a dimerized

peptide with two-proton loss (mass error less than

2.9 ppm), which is presumably an intermolecular

disulfide product formed between two N-terminal

•

studies, using NO as a spin probe and an oxygen

surrogate to form an EPR-visible nitrosyl complex

is a common method to investigate how primary

substrate and dioxygen interact with an EPR-silent

ferrous center.

Initially, we sought to test the binding

•

behavior of NO to Fe^II-ADO. The reduced Fe^II-

ADO protein was completely EPR-silent (Figure

•

6A, dark trace). Addition of 0.1 equiv of NO

releasing agent to the ferrous enzyme resulted in the

formation of a high spin (S = 3/2) resonance with g

values of g_max= 4.10, g_mid= 3.95, and g_min= 2.00

(gray trace). The g value features correspond to an

E/D value ~ 0.01 based on the S = 3/2 rhombogram.

5

Monodentate thiolate coordination of ADO substrates

This nearly axial EPR signal corresponds to the

non-heme ferrous iron center bound to one

non-heme iron-binding proteins (Table S1) (42-46).

The addition of more ^•NO into the sample increased

the DNIC signal, but no {Fe(NO)}⁷(S = 3/2) signal

was observed throughout the addition of ^•NO to the

ES complex. These experiments show that ligation

of the substrate to the iron center increases the

•

equivalent of NO generating {Fe(NO)}⁷, which

can also be described as an antiferromagnetic

coupling between a high-spin Fe^III(S = 5/2) and a

nitroxylate anion (NO^–, S = 1) based on the

Feltham–Enemark theorem (40). Such a high-spin

signal is similar to the {Fe(NO)}⁷species of 3MDO

in the presence of its substrate (8). Additional ^•NO

releasing agent (0.5 and 2.5 equiv) was then added

in the enzyme, resulting in an increase in the signal

intensity of the {Fe(NO)}⁷species (light gray and

red traces). The high-spin signal reached a plateau

•

binding affinity of NO and alters the binding

•

stoichiometry of NO to the Fe. The same DNIC

EPR spectra were obtained from cysteamine-

reduced ADO samples as observed from the

ascorbate-reduced ADO samples upon exposure to

^•NO and the addition of cysteamine (Figure 3F and

Figure S5). Notably, such a dinitrosyl iron complex

was not described in previous EPR studies of other

•

after the addition of 2.5 equiv of NO releasing

•

agent, concomitant with the formation of a minor

thiol dioxygenases. Addition of NO to cysteine-

bound CDO mainly gave rise to a low-spin (S = 1/2),

{Fe(NO)}⁷complex (47), while a high-spin (S =

3/2), {Fe(NO)}⁷complex was generated in the case

of substrate-bound 3MDO (8).

low-spin axial signal with g_┴= 2.039, g_//= 2.015.

The lineshape and the g values of this new S = 1/2

signal are reminiscent of a dinitrosyl species found

in synthetic and protein-based samples (41-43).

However, the dinitrosyl species did not increase

In this work, ADO samples incubated with

^•NO generated either mononitrosyl (S = 3/2) or

dinitrosyl (S = 1/2) complexes, depending on the

existence of the substrate. These complexes

responded differently to temperatures and

microwave power. Therefore, EPR spectra were

recorded under different conditions according to the

power saturation profile (Figure S6).

•

much even when excess NO was added to the

sample, and its population remains insignificant,

accounting for 3% of the total Fe^II-ADO (Figure S3

and Figure S4), indicating that Fe^II-ADO tends to

•

bind only one equiv of NO in the absence of

substrate. The trace amount of dinitrosyl species

•

only forms when excess NO is present, and the

mono-nitrosyl species is predominant in substrate-

free ADO.

Cysteamine monodentate coordinates to ADO

through thiolate ligation

Next, we tested whether the presence of

cysteamine would alter the binding behavior of ^•NO

to the ferrous center. ^•NO was introduced into Fe^II-

ADO precomplexed with cysteamine. The ES

complex was completely EPR-silent (Figure 6B,

black trace). Once a small amount of ^•NO releasing

agent (0.1 equiv) was added, interestingly, no EPR

signal was observed in the high-spin region, rather

a low-spin (S = 1/2) signal was observed with g

values of g_┴= 2.040, g_//= 2.014 (Figure 6B). This

To further investigate the observation that

cysteamine alters the binding behavior of the iron

•

center to NO, cysteamine was titrated into ADO

pre-incubated with excess ^•NO. As shown in Figure

7A and 7B, titration of cysteamine gradually

converted the high-spin {Fe(NO)}⁷complex into

the low-spin {Fe(NO)₂}⁹, which is consistent with

the stoichiometric change of ^•NO from one to two.

Additionally, the newly produced DNIC EPR

signal at g _┴= 2.040, g_//= 2.014 continuously

signal is very similar to the signal from dinitrosyl

species in the aforementioned substrate-free ADO

samples; however, the g values are slightly different.

Also, these two signals responded very differently

increased upon titration of cysteamine (Figure 7B).

The population of DNIC was further quantitated

using Cu-EDTA standard. As shown in Figure S4,

in the presence of saturated cysteamine (30 mM),

the majority of iron centers were converted to

DNIC that accounts for 58% of the total iron

content. Under the circumstance, there is only one

coordination site of iron available for substrate

binding, and the lineshape and g values suggest it is

more likely to have monodentate thiolate ligation to

the iron center (44,48).

•

upon NO titration. This new low-spin signal

produced in the presence of a substrate is more

•

sensitive to the population of NO (Figure 6C).

Compared to similar signals reported in the

literature, it is best explained as a substrate-ligated

dinitrosyl iron center (DNIC), i.e., a B-type

{Fe(NO)₂}⁹species that has been reported in other

6

Monodentate thiolate coordination of ADO substrates

With the understanding that cysteamine

coordinates to the iron center of ADO in a

monodentate fashion, we next attempted to

determine if it is indeed the thiol rather than the

amine moiety of cysteamine that is responsible for

ligation. Substrate analogues, ethanolamine, and β-

mercaptoethanol (β-ME) were added to ADO pre-

complexed with ^•NO. Both analogues are

comprised of a functionalized ethane chain;

ethanolamine replaces the thiol of cysteamine with

a hydroxyl and β-ME replaces the amine with a

hydroxyl (Figure 7C). Substitution of a specific

functional group allows for a detailed comparison

of the binding mode of the organic substrate

through EPR spectroscopy. If the substrate ligates

in a bidentate fashion with both amine and thiol

groups, the EPR spectra of ethanolamine and β-ME

should be distinguishable from the spectrum of

cysteamine. In contrast, if the substrate coordinates

in a monodentate fashion, the EPR spectrum of

cysteamine should have similar spectroscopic

features with either ethanolamine or β-ME based on

which group ligates to iron. Similarity with

ethanolamine or β-ME represents amine or thiol

coordination, respectively.

to the iron center similarly as cysteamine and that

the iron center binds two equiv of ^•NO as well. The

addition of 30 mM cysteamine and β-ME yielded a

comparable population of the {Fe(NO)₂}⁹species,

which shows ADO has a similar affinity to

cysteamine and β-ME at saturating concentrations.

Therefore, the substitution of the amine with a

hydroxyl has

a

negligible impact on the

coordination geometry and electronic structure of

the iron center. Altogether, these data indicate that

cysteamine coordinates to the iron center of ADO

with its thiol group in a monodentate fashion.

RGS5 peptide coordinates to ADO through the N-

terminal thiol

Like cysteamine, the reported N-terminal cysteine-

containing protein substrates of ADO have a free

thiol and amine. However, we wondered if the

peptide chain binds to the iron in the same

monodentate manner. Thus, the N-terminus of

RGS5 peptide was titrated into the enzyme-nitrosyl

complex, and the changes were monitored by EPR

spectroscopy. As shown in Figure 8A, with no

substrate present, the enzyme-nitrosyl complex had

a pronounced high-spin (S = 3/2) {Fe(NO)}⁷signal

(black trace). Under the experimental conditions

(10 K, microwave power 50 mW), an additional

positive absorptive peak was observed at g = 1.98,

Substrate-free ADO exposed to ^•NO had a

predominant high-spin (S = 3/2) {Fe(NO)}⁷and a

minor low-spin (S = 1/2) dinitrosyl species (Figure

6A and Figure S3). The addition of 30 mM

ethanolamine did not cause obvious change to the

EPR spectrum, and the high-spin {Fe(NO)}⁷

remained as the majority of iron signal (Figure 7C,

•

which was assigned to excess free NO in solution

(49). Upon titration of the peptide substrate, the

high-spin signal gradually decreased, consistent

with the observation from the samples which

blue trace). A trace amount of dinitrosyl species (4% underwent cysteamine addition (Figure 8A).

Additionally, the EPR signal of free ^•NO decreased

of total iron) at g_┴= 2.039, g_//= 2.015 was seen in

and completely disappeared upon substrate addition

the spectrum of ethanolamine sample due to the

excess of ^•NO, regardless of the addition of organic

substrate (Figure 7D, blue trace). This result

indicates that ethanolamine has no specific binding

to the metal center of ADO. Alternatively, in the

presence of β-ME, the low-field region became

nearly featureless, while the prominent signal was

a low-spin (S = 1/2) species (Figure 7C, red trace).

The low-spin dinitrosyl species which accounts for

56% of the total iron has nearly identical spectral

features to the {Fe(NO)₂}⁹complex observed when

cysteamine was present (Figure 7D, black and red

traces). The high-spin {Fe(NO)}⁷species without

any organic substrate showed near complete

conversion to the {Fe(NO)₂}⁹complex. The

observation of high-spin to low-spin conversion

upon addition of β-ME indicates that it coordinates

at 5 mM concentration (Figure 8A, red trace). The

•

decrease of free NO signal suggests that the free

^•NO in solution was consumed to form other

nitrosyl species.

Next, the low-spin region of the above

samples was analyzed 50 K. The initial enzyme-

nitrosyl complex had a small dinitrosyl signal at g_┴

= 2.039, g_//= 2.015 (Figure 8B, black trace). As

expected, after titrating the peptide substrate, the

major resonance of the low-spin (S = 3/2),

{Fe(NO)₂}⁹complex shifted to g_┴= 2.040, g_//=

2.014 and intensified with increasing concentration

of the peptide. In the presence of 5 mM RGS5

peptide, the majority of iron center was converted

to DNIC that accounts for 61% of the total iron

7

Monodentate thiolate coordination of ADO substrates

content (Figure S4). This observation mirrors the

phenomenon caused by cysteamine, which suggests

the iron center was coordinated by a thiol group and

from both human ADO and mouse ADO expression

systems. The origin of this absorbance feature

remains unresolved at the present stage. It may

result from a small molecule tightly bound to a

small fraction of protein during protein expression

and cell lysis. Anaerobic addition of ^•NO gave rise

to new, broad peaks centered at 340 and 406 nm

(green trace), which resemble the features observed

in the nitrosyl samples of other non-heme iron

enzymes, including isopenicillin N synthase (IPNS)

catalyzing a thiol-containing substrate (39). Such

absorption bands are attributed to charge transfer

transitions associated with the {Fe(NO)}⁷complex.

The addition of cysteamine resulted in a distinct

peak with a λ_maxat 397 nm, but the peaks observed

in the enzyme-nitrosyl complex were no longer

present (blue trace). The addition of RGS5 peptide

also generated a new peak but with a different λ_max

at 390 nm and more intensity compared to the

cysteamine-bound nitrosyl complex (red trace).

The absorbance bands centered at about 400 nm are

suggestive of thiol-containing mononuclear DNICs

that have been previously documented (50,51).

Therefore, our UV-vis spectroscopic results again

confirm the formation of ADO-based dinitrosyl

complexes in the presence of substrates. The

substrate-bound nitrosyl complex of IPNS had

absorbance features at 508 and 720 nm, which are

quite different from the ADO case since DNIC was

absent in the former. Together with the EPR and

absorbance spectra, the peptide substrate exhibits

similar binding behavior to ADO as cysteamine, but

with higher affinity. Moreover, the thiol-bound,

mononuclear DNIC has been independently

characterized by both spectroscopies.

•

two NO molecules, i.e., the peptide monodentate

coordinates to the iron center via thiolate ligation.

The peptide substrate alters the binding

stoichiometry of ^•NO from one to two equivalents,

as cysteamine does. Based on the EPR spectra, the

DNIC {Fe(NO)₂}⁹signal was more responsive

upon peptide addition than cysteamine addition,

especially at lower concentrations (< 1 mM)

(Figure S4). This suggests the iron center of ADO

has a higher affinity to the peptide substrate than

cysteamine so that the peptide substrate converts

the mononitrosyl complex to the ternary catalytic

mimic, thiol-bound {Fe(NO)₂}⁹complex more

efficiently. Such an interpretation is consistent with

the reported K_mvalues of 71.5 and 3,400 μM for the

peptide and cysteamine, respectively (20,27).

Other than the predominant dinitrosyl peak

at g_┴= 2.040, g_//= 2.014, two new signals were seen

in the low-spin region of peptide samples that were

absent in the samples of cysteamine. An axial signal

arose at g_┴= 2.244, g_//= 2.131 (Figure 8B, marked

by asterisk), and a broad signal centered at g =

2.032 (marked by pound) overlapped with the

major resonance of thiol monodentate bound,

{Fe(NO)₂}⁹complex. Both signals were very

similar to the signals shown in CDO nitrosyl studies,

and the g = 2.032 signal corresponds to a

characteristic, cysteine bidentate bound, ferrous-

nitrosyl {Fe(NO)}⁷complex (28,47). Such an

observation suggests that at high concentrations of

a peptide substrate (at mM level), the amine of the

peptide may interact with the iron center forming

bidentate coordination, but its population is small

compared to the predominant, thiol monodentate

coordination.

Discussion

Among the thiol dioxygenases, CDO has been

unambiguously characterized by substrate

bidentate coordination. Without an ES complex

structure, the substrate-binding mode of 3MDO is

controversial due to the inconsistency between

spectroscopic data and a computational study

(8,38,52), while the substrate binding of ADO and

MSDO remain undefined. Studies on 3MDO and

MSDO consistently suggest an arginine residue

(Arg168 in 3MDO from Pseudomonas aureginosa

and Arg66 in MSDO from Variovorax paradoxus

strain B4) plays a crucial role in their catalysis,

likely interacting through a highly specific salt

bridge to the carboxylate group of substrates, which

As a non-heme iron-dependent enzyme, the

ferrous form of human ADO at the resting state is

expected to be colorless and uncharacteristic above

280 nm by UV-vis spectroscopy. However, the

concentrated protein (200 μM) showed a pale green

color and its absorbance spectrum exhibited a small

peak with a λ_maxat 405 nm (Figure 8C, black trace).

A similar spectral feature is also present in mouse

ADO with λ_maxat 409 nm (Figure S6). This

absorbance feature was only observable in

concentrated protein samples and consistently

showed in samples after multi-step purification and

8

Monodentate thiolate coordination of ADO substrates

correlates to the function of Arg60 in human CDO

(9,52,53). Additionally, SbzM and OvoA are other

two non-heme iron enzymes related to cysteine

oxidation, but yet not classified as thiol

dioxygenases due to their atypical reactions and

different metal ligands. SbzM catalyzes the two-

step oxidation and decarboxylation of cysteine (54),

while OvoA promotes either dioxygenation or

dimerization of cysteine as modulated by histidine

(55). The substrate-binding of neither enzyme has

been spectroscopically characterized. As the only

thiol substrate without a carboxylate group among

these native thiol substrates for this enzyme family

(Figure 1A), lacking the assistance from such an

arginine, cysteamine is likely to bind the enzyme in

a different manner compared to other thiol

dioxygenases. Whether cysteamine binds in a

bidentate fashion with both amine and thiol

coordinated, or as a monodentate ligand with either

its thiol or amine was an open question before this

study. Likewise, substrates of N-terminal cysteine-

containing peptides such as RGS5 contain a free

thiol and amine as well but lack a carboxylate group

(Figure 1B). Determining the specific binding of

these polypeptide substrates in the active site of

ADO is of great importance to further understand

this biologically significant enzyme.

monodentate ligation enables a facile reduction of

the metal center.

Mössbauer

spectra

show

that

approximately 75% of iron center remained as

ferrous iron in as-isolated ADO. Although two

major ferrous species were observed, Fe^II_Aand Fe^II,

B

only Fe^II_Bis directly related to substrate binding and

subsequent catalytic activity. However, there is an

interconversion between these two species

triggered by substrate addition. The mechanism for

ADO preserving such an inhomogenous iron center

is unclear, but it seems to be a common feature in

some non-heme iron dioxygenases besides thiol

dioxygenases (9,31,59,60).

Substrate incubation with oxidized ADO

leads to the finding that the active site of ADO

could be readily reduced by substrate thiol group

and hence reactivate the enzyme if it were to

become oxidized. As a result, substrates form

disulfide by-products, for example, cysteamine

generates cystamine, and RGS5 peptide produces

either intra- or inter-molecular disulfide crosslinks.

Other peptides whose dioxygenation may be

catalyzed by ADO could have different disulfide

products based on the location of cysteine since the

formation of a disulfide bond is a non-enzymatic

outcome that occurs after the activated thiol is

liberated from the active site. Importantly, the

Incubation with either cysteamine or N-

terminus peptide of RGS5 with Fe^III-ADO gave rise

to respective EPR signals, which suggests the

reduction is achieved through direct ligation

between substrates and the enzyme. Similar EPR

signals have been detected in Fe^III-CDO with the

incubation of cysteine, but no cysteine-mediated

reduction was reported (56). Since the thiol groups

of N-terminal cysteine and free cysteine have

similar redox potentials and the iron center of ADO

and CDO have identical coordination (3-His motif),

it is interesting that the substrate-mediated

reduction was only found in the ADO system. A

previous EPR study of CDO pointed out that the

amine group of L-cysteine is likely to coordinate to

the iron center prior to the thiol, and the thiol

coordination is relatively transient in the absence of

^•NO or O₂(47)_,which is also supported by X-ray

absorption and crystallographic data (57,58). A

plausible explanation is that the initial coordination

of amine causes a strong d-orbital splitting of iron,

and thus, resulting in a distinct coordination

geometry that hinders the further reduction by the

thiol. This may explain that in the ADO case, thiol

substrate-mediated reduction could have

a

profound impact on oxygen homeostasis. ADO

regulates N-terminal cysteine-containing proteins

with an extremely high K_mfor oxygen (>500 μM)

(20), which implies that the activity of ADO keeps

a linear relationship with the oxygen concentration

within the physiological range. The reduction effect

of the primary substrates keeps the enzyme ready in

an active form to respond as an oxygen sensor, even

when facing oxidative stress. Such a substrate-

induced reactivation was also reported in a life

essential enzyme, tryptophan 2,3-dioxygenase, that

generates the tryptophan dimer and mono-

oxygenated tryptophan as the by-products (61). The

finding from ADO is another example of how

metalloenzymes maintain their catalytic activity to

defend against an oxidizing environment, such as in

cancer cells. We have previously shown that when

the primary substrate and O₂are both available,

ADO autocatalytically generates a protein-bound

Cys-Tyr cofactor by the iron center that functions

as a catalytic amplifier to more rapidly reduce the

level of the free-thiol bearing substrate (27). Thus,

9

Monodentate thiolate coordination of ADO substrates

the elevated level of the substrate can reactivate

ADO and bring its catalysis up to speed to respond

to the rise of thiol levels.

EPR results with nitrosyl complexes of

ADO have striking differences from other non-

heme iron-dependent enzymes that oxidize thiol-

containing substrates, including CDO, 3MDO, and

resonance, as first proposed by Chasteen and

coworkers (42). The DNIC observed in ADO is a

B-type mononuclear DNIC with thiol-containing

ligands that is commonly described by the formula

{Fe(NO)₂}⁹, with two proposed models as

[Fe³⁺(NO⁻)₂] and [Fe⁺(NO)₂] (64). The former

contains a high-spin (S = 5/2) d⁵-configuration of

IPNS (Table 2). In the absence of substrate cysteine, the iron atom and a triplet state (S = 1) of nitroxyl

the CDO-nitrosyl complex exhibited an S = 3/2,

mononitrosyl {Fe(NO)}⁷EPR spectrum with

similar features as the enzyme-nitrosyl complex of

ADO. The substrate bidentate coordination of CDO

promotes the generation of a broad S = 1/2,

substrate-bound {Fe(NO)}⁷signal. A DNIC was

also seen in CDO as a minor species (approximately

5% of the total iron), which was not a substrate-

bound enzyme-nitrosyl complex (47). In the

absence of a substrate, 3MDO was unable to bind

^•NO, and its substrate coordination mode is unclear.

However, the substrate-bound nitrosyl complex

gave rise to an S = 3/2, {Fe(NO)}⁷species. This

high-spin species showed a very similar EPR

spectrum as the substrate-free nitrosyl complexes of

ADO and CDO, which may imply a unique

substrate binding for 3MDO that differs from both

ADO and CDO. IPNS is a well-documented

enzyme that catalyzes double ring closure of a

tripeptide substrate that monodentate binds to the

iron center via a thiol group (62). Its nitrosyl

complex in the absence of the substrate was

reported to have similar EPR features as ADO and

most non-heme iron-dependent proteins (39). In

contrast to the low-spin (S = 1/2), {Fe(NO)₂}⁹

species of ADO, the ES-nitrosyl complex of IPNS

became more rhombic than substrate-free nitrosyl

complex but remained as a high-spin (S = 3/2),

{Fe(NO)}⁷species. This difference is potentially

due to a different set of first sphere metal-ligand (2-

His-2-carboxylate motif in IPNS). In general, ADO

exhibits a distinct DNIC among these thiol-

oxidizing enzymes.

ligands, while the latter is a high-spin (S = 3/2) d⁷-

configuration of the iron atom bound to two neutral

nitric oxide molecules (S = 1/2). An alternative

model for DNIC is [Fe⁺(NO⁺)₂] with the formula of

{Fe(NO)₂}⁷, which involves the transfer of electron

density from sulfur atoms to iron and nitrosonium

ligands (64). The third model is feasible when a

soluble thiol-bound DNIC is concerned, especially

•

if the DNIC could act as a NO donor and trigger

the S-nitrosation of the thiol. Further investigation

is required to identify the model that best represents

the structure and character of the DNIC present in

ADO.

Presumably, the ES-nitrosyl complex

mimics the ternary structure related to the catalytic

reaction. The observation of a DNIC in ADO in the

presence of primary substrate leads to the

interpretation that both oxygen atoms of molecular

oxygen could spontaneously bind to the iron center

forming an octahedral ternary complex after

monodentate thiolate ligation. If so, our findings

here add a new, side-on oxygen binding model to

the thiol dioxygenase family, since previously

binding modes were exclusively proposed as end-

on (5,8,28,54,58,65). A new oxygen binding mode

potentially represents a new oxygen activation

mechanism during thiol oxidation and Cys-Tyr

crosslink biogenesis in thiol dioxygenases. Our

finding that cysteamine and N-cysteine peptide bind

ADO through thiol group in a monodentate fashion

also supports that the amine group does not

participate in either catalysis or cofactor biogenesis.

•

Moreover, a previous study has shown that NO

The unique phenomenon of DNIC in ADO

affects the N-terminal cysteine oxidation and the

stability of RGS proteins (66), the process that is

regulated by ADO (20). The ADO-nitrosyl

complexes presented here may provide a direction

for investigating how the N-degron pathway acts as

involves the binding of its primary substrate, either

cysteamine or N-cysteine containing peptide. DNIC

could be generated by mononuclear iron, [2Fe-2S],

or [4Fe-4S] cluster, while the mononuclear DNIC

is generally more important in terms of biological

events (63). As summarized in Table S1, despite

the number of the nucleus, the EPR spectra of

DNICs reported in the literature can be classified

into A-type and B-type based on anisotropy of

•

an NO sensor. Hence, even if the ES-nitrosyl

complex is not directly relevant to the catalytic

mechanism, the DNIC formation in ADO certainly

opens a new avenue for correlating ADO with ^•NO

related physiological study.

10

Monodentate thiolate coordination of ADO substrates

Cysteamine (95%), ethanolamine (99%), and 2-

mercaptoethanol (99%) were purchased from Acros

Organics. Peptide substrate with the sequence of

CKGLAALPHSCLER (>95%) was synthesized by

Biomatik, which corresponds to the first 14 amino

acids of the Met-excised N-terminus of RGS5 as

reported previously (20).

Concluding Remarks

Thiol dioxygenases are a family of 3-His

coordinated ferrous enzymes that activate

molecular oxygen and transform thiol substrates to

corresponding sulfinates. Their biological

significance is in regulating sulfur metabolism and

oxygen homeostasis in various organisms.

Although they share some structural and catalytic

features, those enzymes specifically handle

different thiol-containing substrates ranging from

small molecules to N-terminal cysteine-containing

peptides. As the only enzyme catalyzing the

oxidation of substrates without a carboxylate

moiety, the iron center of ADO and its interaction

with cysteamine and RGS5 peptide were

investigated here via biophysical and bioanalytical

techniques, including EPR, Mössbauer, UV-vis

spectroscopies, and mass spectrometry. The results

suggest that ADO preserves an inhomogeneous

ferrous center in the resting state, while only one

form is directly responsible for substrate binding

and subsequent catalysis. Other forms could be

readily converted to the active form in the presence

of primary substrates. We also found a substrate-

mediated reduction of the iron center generating

disulfide by-products. These two features compose

a substrate-induced reactivation mechanism that

enables ADO to function as an oxygen sensor under

physiological conditions. ^•NO, as an oxygen mimic

and spin probe, was incubated anaerobically with

ADO to examine further the binding behavior of

primary substrates and substrate analogues. Our

data suggest that substrates could alter the binding

Cloning, overexpression, and purification

of His₆-tagged, full-length human ADO were

described in our previous publication (27). The

untagged protein was obtained through the cleavage

of His-tag by TEV protease. The tagged and

untagged proteins showed identical activity (k_catof

1.2 s^-1and K_mof 3.4 mM cysteamine) (27) and

spectral features in this study.

The mouse ADO expression plasmid was a

generous gift of Dr. Martha H. Stipanuk (2). Most

of the spectroscopic work, except for Mössbauer

characterization, was carried on both the mouse and

human proteins. No difference was observed unless

otherwise stated. The results presented were mostly

from the human version of the protein. Cell culture

was grown in a baffled flask with Luria Bertani

medium at 220 rpm, 37 °C until OD_{600 nm}reached

0.8 AU. Isopropyl-β-thiogalactoside and ferrous

ammonium sulfate were added to a final

concentration of 600 and 50 μM, respectively,

before the temperature was lowered to 28 °C. After

12 h of further incubation, the cells were harvested

by centrifugation and lysed using a Microfluidizer

LM20 cell disruptor. The enzyme was separated

from the cell lysis using nickel-nitrilotriacetic acid

resin, and was then desalted into 100 mM Tris-HCl

at pH 8.0 and 50 mM NaCl for future use. Protein

concentration was determined by UV-vis

absorbance at 280 nm (ε_{280 nm}= 24,785 cm^-1M^-1)

(27).

•

stoichiometry of NO from one equivalent to two,

generating a thiolate-bound, low-spin, B-type

DNIC signal that is previously unobserved in other

thiol dioxygenases or oxidases. Such an

observation potentially indicates an oxygen side-on

binding mode distinct from other thiol

dioxygenases. The absence of an amine in substrate

coordination also implies that the thiol dioxygenase

promoted oxidation and cofactor biogenesis do not

require the involvement of an amine. Altogether,

our work provides the first systematic investigation

of the ferrous center and substrate binding of ADO,

and it contributes to the overall understanding of the

substrate specificity in thiol dioxygenases.

ADO binds its iron ion tightly. However,

the iron ion in the protein could be easily reduced

by reducing agents such as ascorbate. Hence, the

ferrous form of ADO was prepared by supplying

the iron source during protein expression and then

reducing chemically. The iron occupancy of

purified ADO was determined as approximately 80%

using the published ferrozine method (31).

Enzyme reduction by substrate

The oxidized Fe^III-ADO was prepared by exposing

the as-purified protein in the air at 4 °C in 100 mM

Tris-HCl at pH 8.0, 50 mM NaCl for overnight. An

alternative method to generate oxidized ADO was

Materials and Methods

Chemicals and protein preparation

11

Monodentate thiolate coordination of ADO substrates

to incubate the protein with potassium ferric

cyanide (10 mM) for 5 h before desalting. Proteins

prepared by either air- or chemically oxidized

showed identical spectroscopic features, but human

ADO was less tolerant of chemical reduction. Then

the enzyme was concentrated to 200 μM and

degassed under vacuum. A stock solution of

cysteamine or RGS5 peptide (300 mM) was

prepared in the same buffer and added to the

enzyme to a final concentration of 30 mM under

anaerobic conditions. Samples were incubated in

the glove box, transferred to EPR quartz tubes, and

then frozen in liquid nitrogen. The sample

incubation and handling were controlled within 1,

3, 6, and 10 min, respectively. Spectra were

recorded on a Bruker E560 X-band spectrometer

equipped with a cryogen-free 4 K temperature

system together with an SHQE high-Q resonator

(9.4 GHz). Experimental conditions were set at 100

kHz modulation frequency, 1 mT modulation

amplitude, 0.2 mW microwave power, and a

temperature of 10 K. Each spectrum is the average

of 4 scans.

CH₃CN with 0.1% of HCOOH was used as the

mobile phase. Mass selected for the scan was set at

m/z values of 62, 87, and 153 for positive mode.

Preparation and EPR measurement of nitrosyl

complexes

O₂-free Fe^II-ADO was freshly prepared to generate

nitrosyl complexes. Sodium ascorbate (100 μM)

was included to keep the protein in a reduced

environment. Nitrosyl samples were prepared by

the addition of ^•NO-releasing agent, DEA-

NONOate (≥98%, Cayman Chemical), to Fe^II-

ADO (200 μM) and incubating for 20 min under

•

anaerobic conditions. The amount of NO in each

prepared sample was estimated through titration of

^•NO releasing agent to the enzyme solution (1.5

•

moles of NO can be liberated per mole of DEA

NONOate). For substrate or analogue titration,

small molecules or RGS5 peptide were added to the

enzyme-nitrosyl complex to the concentration of

0.2, 1, 5, or 30 mM. For ^•NO addition experiments,

cysteamine (30 mM) was complexed with Fe^II-

•

ADO (200 μM) prior to the addition of NO-

releasing agent. The samples were transferred to

quartz EPR tubes and slowly frozen in liquid

nitrogen. EPR spectra were recorded on a Bruker

E560 X-band spectrometer equipped with a

cryogen-free 4 K temperature system as described

elsewhere (32-34), with either a dual mode (9.6

GHz, for small molecules) or an SHQE high-Q

resonator (9.4 GHz, for peptide) at 100 kHz

modulation frequency, 0.6 mT modulation

amplitude. High-spin signals were measured at 10

K with a microwave power of 50 mW and one scan

for each spectrum, and low-spin signals were

measured at a higher temperature, 50 K, with a

lower microwave power of 0.05 mW and an

average of four scans for each spectrum. To

visualize both features of high- and low-spin EPR

signals in the same spectrum, the data acquisition

conditions were set to a temperature at 10 K and

microwave power of 1.0 mW.

Mass spectrometry

For RGS5-mediated reduction, the peptide (5 mM)

was anaerobically incubated with oxidized ADO

(200 µM) in NH₄HCO₃(50 mM) at pH 7.0. The

control sample was 5 mM peptide alone in the

buffer. Both samples were incubated at room

temperature for 1 h before removal of the enzyme

using centrifugal filters. The filtrate was then

analyzed by high-resolution mass spectrometry.

Spectra were collected on a maXis plus quadrupole-

time of flight mass spectrometer equipped with an

electrospray ionization source (Bruker Daltonics)

and operated in the positive ionization mode.

Samples were introduced via a syringe pump at a

constant flow rate of 3 μL/min. Important source

parameters are summarized as follows: capillary

voltage, 3,500 V; end plate offset, –500 V;

nebulizer gas pressure, 0.4 bar; dry gas flow rate,

4.0 L/min; source temperature, 200 °C; collision

energy, 5.0 eV. Mass spectra were averages of one

minute of scans collected at a rate of 1 scan per

second in the range 50 ≤ m/z ≤ 1500. For

UV-vis absorbance spectroscopy

The preparation of nitrosyl complexes for UV-vis

absorbance spectroscopic measurement was

identical to the procedure described in the EPR

measurement of nitrosyl complexes. 200 μM, O₂-

free Fe^II-ADO was used in each sample. Excess

^•NO was pre-complexed with enzyme prior to the

addition of 10 mM cysteamine and RGS5 peptide.

cysteamine-mediated reduction, samples were

prepared similarly but with 10 mM cysteamine

anaerobically. The filtrate was injected into LC-MS

for reverse-phase separation after the addition of 50

mM ethanolamine as an internal standard. 50% of

12

Monodentate thiolate coordination of ADO substrates

All spectra were recorded in a 1-cm, anaerobic

quartz cuvette (SpectrEcology) using a Lambda 25

spectrometer (Perkin Elmer).

Data availability

All of the data are described in Tables 1 and 2,

Figures. 1–8 and as well as in the Supporting

Information Table S1 and Figures S1–S6.

Mössbauer spectroscopy

In order to generate ⁵⁷Fe-incorporated ADO with

high metal occupancy, ⁵⁷Fe stock solution was

supplied to the metal-depleted LB medium during

cell culture. The stock solution was prepared by

dissolving ⁵⁷Fe powder (95.4%, Science

Engineering & Education Co.) in 2.5 M H₂SO₄

under anaerobic condition, which was then

anaerobically incubated at 60 °C with continuous

stirring until completely dissolved. The stock

solution of the pH was drop-adjusted by sodium

hydroxide, as described previously (35). A final

concentration of 80 μM ⁵⁷Fe was added for cell

growth. Expression and purification of ADO were

done as aforementioned, and the protein was finally

desalted into a 100 mM Tris-HCl at pH 8.0, 25 mM

NaCl and 5% glycerol buffer. The protein was then

concentrated to 2 - 3 mM. A 500 μL aliquot of the

concentrated protein was transferred into

Mössbauer cup and frozen in liquid nitrogen.

Mössbauer spectra were recorded in a weak-field

spectrometer equipped with a Janis 8DT variable-

temperature cryostat as described previously (35).

The zero velocity of the spectra refers to the

centroid of a room-temperature spectrum of a

metallic iron foil. For samples of the enzyme-

substrate complex, cysteamine was added to a final

concentration of 30 mM under anaerobic conditions.

All spectra were collected at 4.2 K with 50 mT

applied field parallel to γ-radiation. Isomer shifts

are reported relative to Fe metal at 298 K. Least

square fitting of the spectra was performed with the

WMOSS software package (WEB Research, Edina,

MN).

Acknowledgements

This article is dedicated to Dr. Sunil G. Naik, FRSC,

a former postdoctoral associate of this team who

succumbed to H1N1 virus infection in June 2017.

We thank Dr. Martha Stipanuk of Cornell

University for the gift plasmid of mouse ADO and

Dr. Boi Hanh (Vincent) Huynh of Emory

University for access to Mꢀssbauer instrumentation.

We are grateful to Dr. Jiasong Li for helpful

discussion.

Funding and additional information

This work was supported by the National Science

Foundation award CHE-1808637 and in part by the

National Institutes of Health grant GM108988. The

mass spectrometry facility used in this work was

sponsored by the National Institutes of Health

Grant G12MD007591.

Conflict of Interest

The authors declare no competing financial

interests.

ORCID

Yifan Wang: 0000-0003-0378-2469

Ian Davis: 0000-0002-1566-4972

Wendell P. Griffith: 0000-0003-2633-6103

Aimin Liu: 0000-0002-4182-8176

Supporting Information

Multiple sequence alignment, LC-MS, ERP power

saturation, additional EPR and UV-vis data

(Figures S1-S6) and a summary of EPR

parameters of DNICs (Table S1) (PDF).

13

Monodentate thiolate coordination of ADO substrates

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17

Monodentate thiolate coordination of ADO substrates

Figures and Tables

Table 1. Mössbauer parameters of as-isolated and cysteamine-incubated ADO

Γ_L(mm/s)^aΓ_R(mm/s)

Fe species

Fe^III

δ (mm/s)

0.27

ΔE (mm/s)

0.47

% of total iron

23%

0.51

0.34

0.40

0.31

0.40

0.51

0.31

0.34

0.40

0.31

0.43

Fe^II

1.16

2.45

24%

as-isolated ADO

A

Fe^II

1.26

2.93

53%

B

Fe^III

0.27

0.47

14%

cysteamine-

incubated ADO

Fe^II

1.16

2.45

14%

A

Fe^II

1.19

2.85

72%

C

^aSpectra were fitted using Voigt line shape.

18

Monodentate thiolate coordination of ADO substrates

Table 2. EPR and UV-vis absorbance spectroscopic parameters of nitrosyl complexes formed in non-heme iron enzymes catalyzing thiol

oxidation. For EPR parameters, only the enzyme-based, predominant species are summarized here.

Protein ligand

3-His

Substrate coordination

Enzyme-nitrosyl complex

{Fe(NO)}⁷, S = 3/2,

ES-nitrosyl complex

{Fe(NO)₂}⁹, S = 1/2,

g = 2.04, 2.01;

monodentate,

via thiol

ADO¹

g = 4.10, 3.95, 2.00;

340, 406 nm

390 or 397 nm

bidentate,

via thiol and amine

unclear,

{Fe(NO)}⁷, S = 3/2,

{Fe(NO)}⁷, S = 1/2,

g = 2.07, 2.02, 1.98;

{Fe(NO)}⁷, S = 3/2,

g = 4.06, 3.96, 2.00;

{Fe(NO)}⁷, S = 3/2,

g = 4.22, 3.81, 1.99;

508, 720 nm

CDO²

3-His

g ~ 4, 4, 2

3MDO³

N/A

thiol involved

{Fe(NO)}⁷, S = 3/2,

g = 4.09, 3.95, 2.00;

340, 430, 600 nm

2-His-2-

carboxylate

monodentate,

via thiol

IPNS⁴

¹Results from this work.

²Results from references (28,47).

³Results from reference (8,52).

⁴Results from reference (39,62).

19

Monodentate thiolate coordination of ADO substrates

Figure 1. Reactions catalyzed by thiol dioxygenases and superposition of human ADO and

CDO structures. (A) Thiol dioxygenases catalyze the oxidation of thiol-bearing small molecules.

Cysteamine is the only small molecule without a carboxylate group among all the substrates. (B)

Thiol dioxygenases oxidize selective peptides containing cysteine at the N-terminus. (C)

Superposed structures of a predicated model of ADO (green) and a ternary complex of CDO (white,

PDB code: 6N43). The substrate of CDO, cysteine, extensively interacts with the second sphere

residues, including Cys93-Tyr157 crosslink, Arg60, and Tyr58. Cys220-Tyr222 crosslink of ADO

locates at an opposite position compared to CDO.

20

Monodentate thiolate coordination of ADO substrates

Figure 2. Continuous-wave EPR spectra of time-resolved incubation of oxidized human ADO with

(A) cysteamine and (B) N-terminus of RGS5. (black trace) Oxidized Fe^III-ADO exhibited a major high-

spin (S = 5/2) signal centered at g = 4.30 with multiple low-field resonances (inset, amplified by 6 times).

Fe^III-ADO was incubated with 30 mM cysteamine (blue traces) and RGS5 peptide (red traces) for 1, 3, 6,

and 10 min. The EPR signal intensity is present in arbitrary units. Spectra were recorded at 10 K with 0.2

mW microwave power.

21

Monodentate thiolate coordination of ADO substrates

Figure 3. EPR spectra demonstrating cysteamine reduction of the ferric center of mouse ADO. (A)

The freshly prepared, as-isolated mouse ADO, (B) after oxidation by ferricyanide and desalting, (C)

incubation with 50 mM cysteamine for 1.5 min, (D) 5 min after cysteamine incubation, (E) 20 min after

cysteamine incubation, and (F) addition of ^•NO. The signal intensity is present at an arbitrary scale to make

each spectrum in approximately the same height for easy comparison. Spectra were recorded at 10 K with

1.0 mW microwave power 5 G modulation.

22

Monodentate thiolate coordination of ADO substrates

57

Figure 4. Mössbauer spectra of Fe-enriched ADO. (A) As-isolated ADO. Spectrum fitted with three

components, Fe^III(red), Fe^II_A(purple), Fe^II_B(green); (B) ADO with the addition of 30 mM of cysteamine.

Spectrum was fitted to three components, Fe^III(red), Fe^II(purple), Fe^II(blue); (C) The overlay of the

A

c

subtracted spectra of the active enzyme alone (green line) and ES complex (blue line). All spectra were

collected at 4.2 K with 50 mT applied field parallel to γ-radiation.

23

Monodentate thiolate coordination of ADO substrates

Figure 5. Mass spectrometry analysis of RGS5 peptide (A) before and (B) after incubation with

oxidized ADO. Charge states and m/z values of RGS5 N-terminus, intramolecular crosslinked peptide, and

intermolecular crosslinked peptide are marked in black, red, and blue, respectively. The putative structures

and theoretical molecular weight are shown on the right.

24

Monodentate thiolate coordination of ADO substrates

•

Figure 6. EPR spectra of NO addition to ADO in the (A) absence and (B and C) presence of

cysteamine. ^•NO was released by 0 (black), 0.1 (gray), 0.5 (light gray) and 2.5 equiv (red) of ^•NO releasing

agent. (A) In the absence of substrate, changes in the low-field (high-spin) region with increasing NO^•.

Spectra were taken at 10 K with 50 mW microwave power. (B) In the presence of substrate, changes in

both high- and low-spin regions with increasing NO^•. Spectra were taken at 10 K, 1 mW. (C) In the presence

of substrate, changes in the g = 2 region with increasing ^•NO. Spectra were taken at 50 K with 0.05 mW

microwave power.

25

Monodentate thiolate coordination of ADO substrates

Figure 7. The ferrous nitrosyl complex EPR spectra obtained through addition of cysteamine or

analogues to human ADO pre-complexed with excess ^•NO . (A) High-spin and (B) low-spin features of

enzyme-nitrosyl complex after addition of 0 (black), 0.2 (gray), 1 (light gray) and 5 mM (red) cysteamine.

High spin and low spin signals were recorded at 10 K with 50 mW microwave power, and at 50 K with

0.05 mW microwave power, respectively. (C) Full scans and (D) low-spin regions of the enzyme-nitrosyl

complex after the addition of 30 mM cysteamine (black), β-ME (red) and ethanolamine (blue). Structures

of cysteamine, β-ME, and ethanolamine are shown in black, red and blue, respectively. Full scans and low-

spin signals were recorded at 10 K with 1 mW microwave power, and at 50 K with 0.05 mW microwave

power, respectively.

26

Monodentate thiolate coordination of ADO substrates

Figure 8. EPR and absorbance spectra of RGS5 peptide addition to the ferrous form of human ADO

pre-complexed with excess ^•NO. (A) ADO-nitrosyl complex after addition of 0 (black), 0.2 (gray), 1 (light

gray) and 5 mM (red) RGS5 peptide. Spectra were taken at 10 K with 50 mW microwave power. (B) The

g = 2 region of enzyme-nitrosyl complexes after addition of 0 (black), 0.2 (gray), 1 (light gray) and 5 mM

(red) RGS5 peptide. An axial signal (*, amplified by 9 times in inset) and a broad signal (#) were observed.

Spectra were taken at 50 K with 0.05 mW microwave power. (C) UV-vis absorbance spectra of Fe^II-ADO

•

(black), ADO-nitrosyl complex (green), ADO complexed with NO and cysteamine (blue), and ADO

complexed with ^•NO and RGS5 peptide (red).

27

Characterization of the non-heme iron center of cysteamine dioxygenase and its

interaction with substrates

Yifan Wang, Ian Davis, Yan Chen, Sunil G. Naik, Wendell P. Griffith and Aimin Liu

J. Biol. Chem. published online June 28, 2020

Access the most updated version of this article at doi: 10.1074/jbc.RA120.013915

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Characterization of the nonheme iron center of cysteamine dioxygenase and its interaction with substrates

DOI: 10.1074/jbc.ra120.013915

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Article abstract of DOI:10.1074/jbc.ra120.013915

Full text of DOI:10.1074/jbc.ra120.013915

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