A novel catalytic heme cofactor in SfmD with a single thioether bond and abis-His ligand set revealed by ade novocrystal structural and spectroscopic study

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A novel catalytic heme cofactor in SfmD with

a single thioether bond and a bis-His ligand set

revealed by a de novo crystal structural and

spectroscopic study†

Cite this: Chem. Sci., 2021, 12, 3984

All publication charges for this article

have been paid for by the Royal Society

of Chemistry

a

Inchul Shin,

Ian Davis,

Karinel Nieves-Merced,^abYifan Wang,

ab

a

*

Stanton McHardy

and Aimin Liu

SfmD is a heme-dependent enzyme in the biosynthetic pathway of saframycin A. Here, we present a 1.78 A˚

resolution de novo crystal structure of SfmD, which unveils a novel heme cofactor attached to the protein

with an unusual Hx_nHxxxC motif (n ꢀ 38). This heme cofactor is unique in two respects. It contains a single

thioether bond in a cysteine–vinyl link with Cys317, and the ferric heme has two axial protein ligands, i.e.,

His274 and His313. We demonstrated that SfmD heme is catalytically active and can utilize dioxygen and

ascorbate for a single-oxygen insertion into 3-methyl-^L-tyrosine. Catalytic assays using ascorbate

derivatives revealed the functional groups of ascorbate essential to its function as a cosubstrate.

Abolishing the thioether linkage through mutation of Cys317 resulted in catalytically inactive SfmD

variants. EPR and optical data revealed that the heme center undergoes a substantial conformational

change with one axial histidine ligand dissociating from the iron ion in response to substrate 3-methyl-^L-

tyrosine binding or chemical reduction by a reducing agent, such as the cosubstrate ascorbate. The

labile axial ligand was identiﬁed as His274 through redox-linked structural determinations. Together,

Received 20th November 2020

Accepted 21st January 2021

identifying an unusual heme cofactor with

a previously unknown heme-binding motif for

DOI: 10.1039/d0sc06369j

a monooxygenase activity and the structural similarity of SfmD to the members of the heme-based

tryptophan dioxygenase superfamily will broaden understanding of heme chemistry.

rsc.li/chemical-science

heme-based tyrosine hydroxylases (TyrH),⁸including LmbB2

and Orf13.^9–11Such a cofactor dependency for aromatic amino

Introduction

acid oxidation is mechanistically distinct from other types of

tyrosine hydroxylation mediated by nonheme-dependent mon-

ooxygenases such as pterin-dependent tyrosine hydroxylase,^12,13

copper-dependent tyrosinase,¹⁴and the two-component, FAD-

dependent monooxygenase found in SgcC.¹⁵

SfmD catalyzes the regioselective hydroxylation of 3-methyl-L-

tyrosine (3-Me-^L-Tyr) to produce 3-hydroxy-5-methyl-L-tyrosine

(3-OH-5-Me-^L-Tyr) in Streptomyces lavendulae (Fig. 1A). Down-

stream enzymes further process the reaction product into the

core quinone structure of saframycin A,^1,2

a

tetrahy-

SfmD was previously reported as a heme peroxidase with an

atypical peroxidase activity of hydroxylating 3-Me-^L-Tyr or L-Tyr

using hydrogen peroxide as oxidant.²To date, SfmD has been

classied in various protein databases as a h member (EC

1.11.2.5) of heme-dependent peroxygenases (EC 1.11.2) in

which one O-atom from hydrogen peroxide is incorporated into

3-Me-^L-Tyr. However, both peroxidase and peroxygenase classi-

cations are problematic for SfmD. Peroxidase enzymes (EC

1.11.1) generally catalyze one-electron oxidation of their

substrate, whereas peroxygenases typically utilize a thiolate-

ligated heme as their catalytic cofactor. In contrast, SfmD

performs monooxygenation and is anticipated to have a histi-

dine-ligated heme in its active site.

droisoquinoline alkaloid with demonstrated antitumor and

antimicrobial activities.³Other saframycin analogs, such as

Ecteinascidin (ET-743) and Zalypsis, are approved drugs. The

former treats so tissue sarcoma,⁴and the latter has undergone

clinical trials for the treatment of Ewing's sarcoma.^5,6Due to its

potential pharmaceutical application, the synthetic pathway of

saframycin A has garnered attention to the unique pentacyclic

tetrahydroisoquinoline scaﬀold built upon ^L-tyrosine (L-Tyr).⁷

SfmD requires a mononuclear heme as its prosthetic group for

oxygenation, similar to enzymes with analogous activity, i.e.,

^aDepartment of Chemistry, The University of Texas at San Antonio, One UTSA Circle,

Texas 78249, USA. E-mail: Feradical@utsa.edu

^bCenter for Innovative Drug Discovery, The University of Texas at San Antonio, One

Peroxidases and peroxygenases are among the most well-

known heme-containing enzyme groups along with cyto-

chrome P450 enzymes and a dioxygenase superfamily named

UTSA Circle, Texas 78249, USA

† Electronic supplementary information (ESI) available. See DOI:

10.1039/d0sc06369j

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Fig. 1 SfmD is an ascorbate- and O₂-dependent monooxygenase. (A) The catalytic reaction of SfmD, (B) HPLC analysis of the hydroxylation of 3-

Me-^L-Tyr catalyzed by SfmD. Absorbance at 280 nm was monitored and presented with the same scale. Peaks in the red and blue shaded boxes

correspond to substrate and product, respectively. The reaction conditions were (i) 3-Me-^L-Tyr, (ii) SfmD and 3-Me-L-Tyr, (iii) SfmD, 3-Me-L-Tyr,

and H₂O₂, (iv) SfmD, 3-Me-L-Tyr, H₂O₂, and ascorbate, (v) SfmD, 3-Me-L-Tyr, and ascorbate under anaerobic condition, (vi) SfmD, 3-Me-L-Tyr,

and ascorbate under air-saturated conditions, (vii) SfmD, 3-Me-^L-Tyr, and ascorbate under O₂-saturation. The concentrations were: 30 mM

enzyme, 1 mM substrate 3-Me-L-Tyr, 1 mM H₂O₂, and 20 mM ascorbate, (C) the maximum absorbance (l_max) of the substrate 3-Me-L-Tyr (red

color trace) and reaction product 3-OH-5-Me-L-Tyr (blue), and (D) positive-mode ESI mass spectra for the enzyme reaction product. Mass

spectra are shown in range 211 # m/z # 216. Experimental mass of 3-OH-5-Me-^L-Tyr matches the calculated one with 0.47 ppm mass accuracy.

aer tryptophan 2,3-dioxygenase (TDO). The iron ion in the suggested to be a new member of the TDO superfamily; however,

heme prosthetic group in these enzymes is bound to a protein- this assignment proposal is yet to be structurally established.¹⁹

based proximal ligand. Cytochromes P450 and peroxygenases

Based on our structure prediction using the primary

have a cysteine-ligated heme, while peroxidases and TDO sequence by Phyre2 server (ESI†) along with the structural

superfamily enzymes employ a histidine-ligated heme. The homolog search by HHpred in the previous work,²SfmD is ex-

impact of the proximal ligand preference, especially cysteine vs. pected to possess structural homology with TDO and its sibling

histidine to the heme orientation and chemistry, has been an enzyme IDO. Intriguingly, IDO and TDO are the predominant

intriguing topic for heme chemistry and biology.¹⁶It is generally members of the heme-containing dioxygenases, which activate

regarded that thiolate ligation of the heme iron enhances dioxygen and catalyze stepwise O-atom incorporations into

hydrogen-atom-abstraction and O–O bond heterolysis, whereas tryptophan via epoxyindole and a ferryl intermediate.^20–24The

histidine ligation favors 1e^ꢁoxidations, or storage and transfer predicted structural relation of SfmD to IDO and TDO, rather

of diatomic gases.¹⁷

than peroxidases and peroxygenases, prompted us to consider

The TDO superfamily occupies a unique position in heme- whether SfmD can catalyze its monooxygenation reaction with

based enzymes because dioxygenation reactions are typically dioxygen and an additional electron source. If SfmD could

catalyzed by non-heme metalloenzymes. The TDO superfamily is catalyze the monooxygenation reaction while reducing dioxygen

a small, structurally related group of proteins, with only four to a water molecule, other than the primary substrate 3-Me-^L-Tyr

identied members thus far, i.e., two indoleamine 2,3-dioxyge- that provides two of the four electrons for dioxygen reduction

nases (IDO) proteins IDO1 and IDO2, TDO, and PrnB in the during catalysis, a cosubstrate must provide 2e^ꢁand 2H⁺to

pyrrolnitrin biosynthesis pathway.¹⁸The substrate of these balance the reaction.

enzymes is tryptophan or a derivative, i.e., 7-chlorotryptophan

In the present work, we chemically synthesized the substrate

for PrnB. Recently, a tryptophan monooxygenase, MarE, is of SfmD, conducted enzyme assays under various conditions,

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and performed spectroscopic and structural characterizations electron donors. The reaction mixture containing the enzyme,

of SfmD. We demonstrated the ability of SfmD to utilize substrate 3-Me-^L-Tyr, and ascorbate (also commonly known as

molecular oxygen as an oxidant and identied ascorbate as the vitamin C) was found to yield the same product under aerobic

source for protons and additional electrons for dioxygen conditions, which eluted at 4.3 min (Fig. 1B). This reaction

reduction in vitro. Moreover, we report the de novo crystal product showed the spectral characteristic of 3-OH-5-Me-^L-Tyr

structure of SfmD solved by multiple anomalous dispersion (Fig. 1C). High-resolution mass spectrometry analysis of the

(MAD) with seleno-^L-methionine (SeMet) incorporation. This product revealed an m/z of 212.0916 with 0.47 ppm of mass

structure is the rst of its kind in a novel class of heme- accuracy, which is a 16 Da increase over that of 3-Me-^L-Tyr and

dependent monooxygenase enzymes. It reveals a unique pros- consistent with expected hydroxylation (Fig. 1D). To further

thetic heme cofactor not previously found in any other enzymes. explore if the reaction is an O₂-dependent process, the assay was

The structural study unveils that the overall structure of SfmD is executed under three conditions: anaerobic, aerobic, and O₂-

reminiscent of tryptophan dioxygenase, and its catalytic heme saturated conditions. Under anaerobic conditions, 3-Me-^L-Tyr

center has a single thioether covalent attachment with a bis-His remained unchanged, and no reaction was observed. When

ligand set in a novel heme-binding motif.

using an air-saturated buﬀer, we observed the conversion of 3-

Me-^L-Tyr to the oxygenated product. Running the reaction using

an O₂-saturated buﬀer led to an accelerated and complete

transformation of 3-Me-^L-Tyr to 3-OH-5-Me-L-Tyr, indicating

that the SfmD reaction is an O₂-dependent process (Fig. 1B). L-

Tyr is a less eﬀective substrate as compared to 3-Me-^L-Tyr.²

Results

The full-length tag-free SfmD is capable of activating dioxygen

for monooxygenation

The native substrate of SfmD, 3-Me-^L-Tyr, was synthesized and When the alternate substrate was tested, the O₂- and ascorbate-

characterized by mass spectrometry and ¹H NMR spectroscopy dependent monooxygenation of ^L-Tyr led to L-DOPA production

(ESI Fig. S1†). It was converted to the Mosher's ester for the (Fig. S3†).

examination of the stereochemical conguration, and the NMR

Next, we recruited catalase to the reaction mixture to remove

data suggest a single ^L-enantiomer. Full-length and truncated any unexpected H₂O₂production from O₂by the enzyme. In

(16–353) forms of SfmD with an N-terminal cleavable His-tag a parallel control experiment, the reaction mixture of SfmD, 3-

were constructed and codon-optimized. The isolated SfmD Me-^L-Tyr, and catalase (10 mg) failed to produce 3-OH-5-Me-L-Tyr

proteins were subsequently subjected to enzymatic assays and (or ^L-DOPA) with added H₂O₂as the oxidant, indicating that the

structural characterizations. Since the C-terminus is predicted catalase was suﬃcient and eﬀective. In contrast, the catalase-

to contain the catalytic center, we constructed an expression containing reaction mixture with ascorbate, but not H₂O₂,

system of the full-length SfmD with an N-terminal polyhistidine successfully generated the product under aerobic conditions

tag followed by a protease cleavage site (see ESI†). A typical (Fig. S4A†). The same observation was obtained for the alternate

catalytic assay with 3-Me-^L-Tyr using full-length, tag-free, SfmD substrate ^L-Tyr (Fig. S4B†). No monooxygenation product or

as the catalyst to form 3-OH-5-Me-^L-Tyr is shown in Fig. 1. SfmD disappearance of 3-Me-^L-Tyr (or L-Tyr) was observed before the

was conrmed to catalyze the monooxygenation of 3-Me-^L-Tyr addition of ascorbate. These results exclude the possibility that

using hydrogen peroxide as the oxidant, as previously shown for H₂O₂was generated from O₂in situ and subsequently used as

this enzyme with a C-terminal His-tag.²A reaction mixture the oxidant for the hydroxylation reaction. In the presence of

containing enzyme, substrate, and hydrogen peroxide produced ascorbate, the ferric heme in SfmD was reduced to ferrous. We

the hydroxylated product, 3-OH-5-Me-^L-Tyr, which eluted at have found that the ferrous form of SfmD was unable to

4.3 min with a spectral maximum centered at 279 nm, a 3 nm produce the oxygenated product from H₂O₂, either under

diﬀerence from 3-Me-^L-Tyr, which is centered at 276 nm. aerobic or anaerobic conditions (Fig. S5†). Kinetic assay of

Subsequent kinetic assays were conducted using N-terminal SfmD was carried out w_ꢃith ^L-Tyr, ascorbate, and O₂-saturated

His-tagged full-length SfmD on the alternate substrate ^L-Tyr buﬀer at pH 9.0 and 25 C (Fig. S2†). Kinetic parameters were

and H₂O₂by monitoring the formation of 3,4-dihydroxy-L- obtained from the hyperbolic curve tting were K_M¼ 1.8 ꢂ

phenylalanine (^L-DOPA) by HPLC. The activity of SfmD was not 0.1 mM, k_cat¼ 0.029 ꢂ 1 min^ꢁ1, and k_cat/K_M¼ 0.016 ꢂ 0.002

well-described by Michaelis–Menten kinetics, and tting the mM^ꢁ1min^ꢁ1. Although these are over 1000-fold less than that

data with a hyperbolic function, as done in a previous study, with H₂O₂, the concentration of O₂in cells is between 200–300

resulted in a poor R²value. The sigmoidal Hill equation yielded mM that is typically more than three orders of magnitude higher

a superior t and the following kinetic parameters: n value of than the H₂O₂level. Thus, the above experiments show that

2.5, K_half¼ 1.8 ꢂ 0.1 mM, k_cat¼ 32.4 ꢂ 1.6 min^ꢁ1, and k_cat/K_halfSfmD can catalyze an O₂-dependent monooxygenation reaction

¼ 18.1 ꢂ 1.9 mM^ꢁ1min^ꢁ1(Fig. S2†), where K_halfis the substrate using ascorbate as a cosubstrate.

concentration at half of the maximal velocity. The origin of the

positive cooperativity for substrate binding to the protein

remains to be resolved in future studies; however, the overall

catalytic performance is comparable with the previous inde-

Ascorbate is a slow reducing agent of the ferric heme in SfmD

but specic as a cosubstrate

pendent study using a C-terminal His-tagged version of SfmD.²The electronic absorption data of SfmD showed that ascorbate

To examine if the enzyme can utilize O₂as the source of slowly reduces the ferric heme compared to the reduction by

oxygen for hydroxylation, we conducted tests with various dithionite within 3 min. The addition of ascorbate to SfmD

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Fig. 2 Ascorbate slowly reduces SfmD. (A) UV-vis spectra of SfmD (black trace) and SfmD with ascorbate (blue trace), (B) the same as (A) except

with the presence of 3-Me-L-Tyr, (C) UV-vis spectra of SfmD with ascorbate (black trace) and SfmD with ascorbate and cNO (blue trace), and (D)

the same as (C) except with the presence of 3-Me-^L-Tyr. The plots for changes at 425 nm as a function of time are shown in the inset in panels

(A)–(D).

under anaerobic conditions resulted in a red-shi of the Soret the reaction of xanthine with xanthine oxidase. The ratio of

band, from 410 to 425 nm with increased absorbance intensity oxidized and reduced forms in terms of protein and dye was

(Fig. 2A). Additionally, the a/b region became more pronounced obtained from the resulting spectra. Aer the linear tting of

at 528 and 558 nm. Plotting the absorbance increase at 425 nm data to the Nernst equation, the reduction potential of SfmD

as a function of time yielded the reduction kinetics in two was calculated to be ꢁ130 ꢂ 1 mV under the specied experi-

phases with the determined reciprocal relaxation times for fast mental conditions (Fig. S8†). As will be discussed later, this is an

and slow. The reciprocal relaxation times are summarized in apparent E_mvalue (E^a_m^pp), which represents a non-canonical

Table S1.† This observation suggests a protein conformational Fe(^II)/Fe(III) couple as the heme center at these redox states

diﬀerence between the two oxidation states of the heme. The possesses distinct ligand sets and protein environment.

substrate binding to SfmD led to a blue-shi of the Soret from

The next question to address is whether ascorbate is

410 to 405 nm (Fig. 2B). The addition of ascorbate to the a specic cosubstrate of the reaction or merely an electron

complex of ferric SfmD with 3-Me-^L-Tyr showed a similar two- source to reduce ferric heme. Several reducing agents were

phase reduction pattern (Fig. 2B). With ^L-Tyr, the alternate investigated for their ability to promote the SfmD reaction,

substrate binding to the active site resulted in the blue-shi of including dithionite, dithiothreitol, b-mercaptoethanol, and

the Soret from 410 to 409 nm (Fig. S6†). Likewise, the addition tris(2-carboxyethyl)phosphine (TCEP), up to 20 mM concentra-

of ascorbate to the binary complex with ^L-Tyr showed a similar tion. These reducing agents could reduce the ferric heme

pattern of two-phase reduction. The addition of cNO to SfmD (Fig. S9†). However, unlike ascorbate, these reducing agents

with ascorbate (Fig. 2C), SfmD and 3-Me-^L-Tyr with ascorbate were unable to sustain the reaction and form a detectable

(Fig. 2D), and SfmD with ^L-Tyr and ascorbate (Fig. S7†) all amount of product, except for dithionite, which generated

showed similar spectral signature changes with a blue-shi of a trace amount of the monooxygenated product that was not

the Soret from 425 to 413 nm and a decrease at 528 and 558 nm catalytic and only accounted for approximately one turnover

in the a/b region. The reciprocal relaxation times were deter- (Fig. S10†). In the oxygen-dependence experiment, it is note-

mined using the reduction at 425 nm (Table S1†).

worthy that a decrease of ascorbate was observed, which had

To evaluate the redox potential of SfmD, we measured the a retention time around 1.7 min, as the hydroxylation product

midpoint Fe²⁺/Fe³⁺reduction potential of SfmD by a dye- accumulated. In the ascorbate-containing reaction mixture, the

coupled assay (see ESI†). In the coupled assay, Nile Blue and peak at 1.1 min was more pronounced in the fully converted

SfmD were reduced at the same time by electrons provided from system (Fig. 1B), which was likely a degradation product of

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ascorbate. Together, the results suggest that ascorbate is hydroxo substituted, 3-O-ethyl-^L-ascorbate showed a small

a cosubstrate of the reaction, providing both electrons and amount of product formation, approximately a single-turnover.

protons during catalysis.

Thus, the hydroxyl group at C3 is important for the cosubstrate.

We further tested various ascorbate derivatives to examine The eﬀects of the other three compounds on product formation

the cosubstrate specicity and probe which functional groups were negligible. The results suggested that the integrity of

of ascorbate are crucial to sustaining the SfmD reaction. In this ascorbate's chemical structure is critical for functioning as

set of experiments, 3-O-ethyl-^L-ascorbate, 2-O-a-D-glucopyr- a cosubstrate. The oxidation state of ascorbate is also essential,

anosyl-^L-ascorbate, 5,6-isopropylidene-L-ascorbate, L-ascorbate as the oxidized form, ^L-dehydroascorbate, failed to function as

2-phosphate, and the oxidized form of ascorbate, ^L-dehy- a cosubstrate. Since ascorbate functions as both an initial

droascorbate were examined (Fig. 3A). Among them, only 5,6- reducing agent when ferric heme was used and a cosubstrate to

isopropylidene-^L-ascorbate showed a nearly comparable reac- sustain the reaction catalytically, it became necessary to

tivity to ascorbate, indicating the C5 and C6 hydroxyl groups are examine whether the non-reactivity of those derivatives was due

insignicant for ascorbate in the SfmD reaction (Fig. 3B). The 3- the inability of iron reduction to the ferrous state. Therefore, we

Fig. 3 Investigation of the structure-based speciﬁcity for ascorbate as a cosubstrate during catalysis. (A) Ascorbate derivatives were employed in

this study, (B) HPLC analysis of the hydroxylation of ^L-Tyr catalyzed by SfmD. Absorbance at 280 nm was monitored and presented with the same

scale. Peaks in the red and blue shaded boxes correspond to substrate and product, respectively. The reaction conditions were (i) ^L-Tyr, (ii) SfmD,

L-Tyr, and ascorbate, (iii) SfmD, L-Tyr, and 3-O-ethyl-L-ascorbate, (iv) SfmD, L-Tyr, and 2-O-a-D-glucopyranosyl-L-ascorbate, (v) SfmD, L-Tyr,

and 5,6-isopropylidene-L-ascorbate, (vi) SfmD, L-Tyr, and L-dehydroascorbate, (vii) SfmD, L-Tyr, and L-ascorbate 2-phosphate, (C) the same as (B)

except executed reaction with ferrous SfmD. The concentrations were: 30 mM enzyme, 1 mM L-Tyr, and 20 mM ascorbate and its derivatives.

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˚

conducted the above experiments using SfmD with the ferrous, a 2.00 A resolution structure of the SfmD variant was deter-

instead of ferric, heme cofactor (Fig. 3C). The results showed mined using non-labeled protein. Finally, we determined a 1.78

˚

a similar pattern with the ferric heme enzyme, indicating that A resolution crystal structure of the full-length SfmD (Table 1).

a structural incompatibility causes the lack of reaction with However, both N- and C-terminal residues (1–15 and 327–365)

ascorbate analogs. We also examined if the analogs can reduce were found to be disordered in the full-length protein structure.

the ferric heme by UV-vis spectroscopy. The reduction of SfmD The superposition of the full-length crystal structure and the

˚

by 5,6-isopropylidene-^L-ascorbate showed a similar pattern with truncated variant shows a 0.25 A root-mean-square deviation

ascorbate, but its rate decreased by ꢀ2-fold compared to (rmsd) for 303 C_aatoms. Therefore, no signicant diﬀerence

ascorbate (Fig. S11 and Table S1†). Other than 5,6-O-iso- between the core structures was observed.

propylidene-^L-ascorbate, no signicant spectral changes were

The SfmD crystal belongs to the P3₁21 space group and

observed within 20 min. These observations revealed their contains one SfmD monomer in an asymmetric unit. The

inability to reduce SfmD to the catalytically active form with O₂. overall crystal structure of SfmD consists of 11 a-helices, 2 short

b-sheets, and one short 3₁₀-helix (h1) (Fig. 4A). The structure

can be described as two layers: an N-terminal half which

includes b1-h1-a1–a4-b2-a5-a6, and a C-terminal half which

covers a7–a11. The two layers face each other with a buried

The three-dimensional structures of SfmD reveal that the

catalytic machinery is an unusual covalently linked heme

prosthetic group with a previously unknown binding motif

2

25

˚

surface area of 2396 A , as calculated by PISA. Extra electron

density was found near the C-terminus and assigned as a heme

cofactor. The ve consecutive a-helices ranging from a7 to a11

in the C-terminus hold the heme prosthetic group, and the rest

of the N-terminal half including b1-h1-a1–a4-b2-a5-a6 is situ-

ated adjacent to the core helical bundles (a7–a11) holding the

prosthetic group. The ve consecutive C-terminal helices (a7–

a11) wind upwards counterclockwise, forming a heme-binding

To facilitate crystallization and structural determination of

SfmD, we constructed a truncated variant containing residues

16–353. We obtained crystals of the variant through the hanging

drop vapor diﬀusion method. However, no existing structures,

including those from IDO/TDO could be used as a search probe

to determine SfmD structure by molecular replacement. Thus,

we prepared seleno-^L-methionine (SeMet)-substituted SfmD,

˚

and determined a 2.20 A resolution crystal structure of the

SeMet-substituted SfmD variant by MAD (Table S2†). Next,

Table 1 X-Ray data collection and reﬁnement statistics

Partially reduced

Truncated

6VDP

Full-length (black in Fig. 6A)

Partially reduced (blue in Fig. 6A)

6VDZ

(green in Fig. 6A)

PDB code

6VDQ

6VE0

Data collection

Space group

P3₁21

Cell dimensions

˚

a ¼ b, c (A)

59.9, 155.9

59.9, 155.8

90, 90, 120

50.00–1.78 (2.23–1.78)

6.5 (88.0)

49.0 (2.2)

59.9, 160.7

90, 90, 120

50.00–2.95 (3.00–2.95)

8.2 (151.0)

59.5, 160.0

90, 90, 120

50.00–3.15 (3.20–3.15)

10.3 (108.4)

22.6 (2.0)

a, b, g (^ꢃ)

Resolution (A)

R_symor R_merge(%)

I/sI

90, 90, 120

50.00–2.00 (2.07–2.00)^a

9.5 (168.9)

˚

36.0 (1.8)

26.3 (1.3)

Completeness (%)

Redundancy

CC_1/2

99.9 (99.9)

19.2 (17.7)

0.996 (0.753)

99.9 (100.0)

12.6 (12.5)

0.994 (0.911)

99.0 (99.5)

7.8 (8.0)

0.994 (0.919)

99.9 (100.0)

9.4 (8.6)

0.997 (0.744)

Renement

Resolution (A)

˚

49.19–2.00

22 614

19.63/24.36

49.22–1.78

31 878

19.92/22.80

49.36–2.95

7411

25.99/32.54

49.01–3.15

6117

26.04/30.77

No. reections

R_work/R_free(%)

No. atoms

Protein

2254

43

2279

43

2241

43

2241

43

Heme

Water

B-factors (A )

74

171

0

2

˚

Protein

Heme

Water

46.1

43.4

47.3

39.4

37.9

46.4

106.8

111.8

N/A

110.4

113.0

N/A

R.m.s. deviations

˚

Bond lengths (A)

0.007

0.880

0.007

0.867

0.002

0.582

0.004

0.681

Bond angles (^ꢃ)

a

Values in parentheses are for the highest resolution shell.

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Fig. 4 The three-dimensional structure of SfmD showing a novel heme cofactor and the heme-binding motif. (A) The overall structure of SfmD

(PDB entry: 6VDQ) is shown in two diﬀerent orientations. Helices in the N-terminal and C-terminal half are presented in blue and white,

respectively. Heme is shown in the red space-ﬁlling model, (B) A single cysteine–vinyl thioether bond, and a bis-His axial metal–ligand motif

revealed a novel heme prosthetic group. Cys317 covalently connects to the heme, whereas Cys234 does not. (C) 2F_oꢁ F_celectron density maps

from the ﬁnal structural reﬁnement are presented in blue contoured at 1s. Omit F_oꢁ F_celectron density maps for heme and Cys317 after

simulated annealing are shown in orange contoured at 3s. (D) Amino acid sequence alignment of SfmD and the hypothetical SfmD-like proteins

is shown with the secondary structural elements of SfmD. Asterisks represent the residues involved in heme binding: His274, His313, and Cys317

in SfmD. (E) Superposition of the core structures of SfmD with TDO (PDB entry: 2NOX). SfmD is shown in white and cyan color for helices and

loops with a heme (pink) bound. TDO is presented in blue and orange color for helices and salmon color for loops with a heme (red). Orange

helices are the secondary structural elements not present in SfmD. Parentheses denote the secondary structure elements of TDO.

˚

pocket where the heme is inserted perpendicular to the plane of and 2.3 A, respectively. One heme 4-vinyl group is covalently

the layer (Fig. 4A).

bound to Cys317 from the last loop aer a11 (Fig. 4C and S12†).

One salient feature of the SfmD crystal structure is its pros- The distance between Cys317 and the heme 4-vinyl group is 1.8

˚

thetic group. Unlike the typical heme c attachment in a CxxCH A, indicating a cysteine–vinyl link with a thioether bond

pentapeptide segment,²⁶the heme bound to SfmD is identied between the heme and the protein. We noticed that the two

as a single cysteine linked c-type heme but with a novel binding axial ligands, His274 and His313, and the thioether-crosslink

motif of Hx_nHxxxC (n ꢀ 38). Another unusual feature of SfmD is contributor, Cys317, in the binding motif of Hx_nHxxxC are all

a bis-histidine (bis-His) ligated set found in both full-length and strictly conserved across the SfmD homologs (Fig. 4D).

truncated SfmD (Fig. 4B). His274 from a9 and His313 from a11 However, the number of residues between the two histidine

˚

form axial ligands for the heme iron with bond lengths of 2.2 A residues (n ꢀ 38) shows relaxed conservation. It varies from 37

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to 40 within the SfmD-like proteins due to deletion or insertion the distal pocket; however, it points away from Trp271 and

in some protein sequences (Fig. 4D). A second cysteine residue, His274, opposite to the heme center. Such a spatial arrange-

Cys234, is also present in the active site, but it is not a conserved ment, together with less pronounced peroxidase activity, further

amino acid across SfmD-like proteins (Fig. 4D). Cys234 is supports that peroxidase activity is not the primary function of

˚

located 4.9 A away from the nearest vinyl group of the heme. SfmD. Rather, SfmD is a structural homolog of the TDO

Thus, Cys234 is not covalently linked to the prosthetic group superfamily with the capability of dioxygen activation for

(Fig. 4B and C). Therefore, the de novo structural data reveal an monooxygenation.

unprecedented singly covalently attached prosthetic group with

a new binding motif and novel covalency.

One axial heme ligand rotates out upon substrate binding or

chemical reduction of the heme iron

The crystal structure of SfmD presented in this work enabled

a structural alignment using the DALI server.²⁷The structurally

ordered region (18–321) of SfmD fetched TDO from Xanthomonas

campestris (XcTDO, PDB code-chain id, 3BK9-B) as the best match

with a Z-score of 7.0, sequence identity of 7%, and an rmsd value

We characterized the SfmD and substrate interactions by EPR

spectroscopy. The crystal structure revealed that the heme

center has a bis-His axial ligand set, which should give rise to

a low-spin ferric heme signal. However, the two axial ligand

planes are nearly perpendicular, which leads to a so-called

highly anisotropic low-spin (HALS) rhombic EPR envelope

frequently seen in many bis-His coordinated hemes.³¹It is

a well-established phenomenon that the EPR signals of such

low-spin ferric heme (S ¼ 1/2) are broadened to the extent that

they are nearly invisible.^31,32Indeed, the covalently bound low-

spin ferric heme in SfmD was practically unobservable by the

low-temperature EPR spectroscopy (Fig. 5A). However, a high-

spin ferric heme EPR signal (S ¼ 5/2), typical of a His-ligated

heme and accountable for all the ferric iron, emerged upon

the addition of 3-Me-^L-Tyr (Fig. 5A). This observation suggests

that one of the axial histidine ligands dissociates from the

iron(^III) center upon substrate binding.

˚

of 2.9 A (for details, see ESI†). CmTDO aligned well with the C-

˚

terminus (residues 180–321) of SfmD with an rmsd of 2.26 A

for 102 C_acarbons (Fig. 4E). As anticipated by structural predic-

tion, the overall structure of SfmD resembles the core structure of

TDO. The secondary structural elements in the C-terminal half of

the SfmD superimpose with TDO (Fig. 4E). There are also

signicant diﬀerences between these two structures caused by

the low sequence identity of 10.5%. Notably, the heme prosthetic

groups of the two enzymes do not align (Fig. 4E). The SfmD

structure also lacks additional secondary structural elements

present in TDO (orange helices labeled as h3 and a7 in Fig. 4E).

The DALI search for the N-terminal half of SfmD (18–181) only

˚

showed similarity to IpaD (2JAA-B; 5.9; 8%; 4.2 A), a Type III

secretion system needle tip protein,²⁸and two synthetic proteins

We further investigated whether one or both histidine resi-

dues ligate the heme iron when in the ferrous oxidation state in

the absence and presence of the primary substrate, 3-Me-^L-Tyr.

Nitric oxide (cNO) was employed as a spin probe and O₂surro-

gate generated through a nitric oxide-releasing agent. The

addition of ascorbate to both SfmD and SfmD complexed with

3-Me-^L-Tyr decreased the high-spin ferric EPR signal, indicating

the chemical reduction of SfmD and the enzyme–substrate

complex by ascorbate to the ferrous oxidation state (Fig. S14†).

The subsequent introduction of cNO under anaerobic condi-

tions led to a low-spin (S ¼ 1/2) EPR signal characteristic of

a His-ligated ferrous heme–nitrosyl complex (Fig. 5B). Thus,

˚

(6FES-A; 6.1; 8%; 3.7 A and 3U3B-B; 5.9; 5%; 2.9 A).

Notably, SfmD was previously dened as a heme peroxidase,

even though its function is monooxygenation.²We executed an

assay using 2,2⁰-azino-bis(3-ethylbenzothiazoline-6-sulfonic

acid) (ABTS), a common benchmark peroxidase substrate, for

assessing its peroxidase activity. SfmD showed a very weak

peroxidase activity (Fig. S13†), which is weaker than that of

a recently characterized cytochrome P450 enzyme CYP121.²⁹In

peroxidases, a distal arginine residue is critical for the forma-

tion of compound I and controlling its reactivity and stability by

H-bond interaction.³⁰In SfmD, Arg272 is adjacent to Trp271 in

Fig. 5 A distal heme ligand of SfmD swings oﬀ from the iron ion in response to substrate binding or chemical reduction. (A) EPR spectra of SfmD

(200 mM, black trace) and SfmD after addition of 3-Me-L-Tyr (2.5 mM, red trace), (B) EPR spectra of ferric SfmD (gray trace), after addition of

ascorbate and cNO (black), and after addition of 3-Me-L-Tyr, ascorbate, and cNO (blue). The EPR spectra of the ferric SfmD were measured at 10

K, while the nitrosyl complexes were measured at 50 K (see ESI,† Materials and methods).

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only one His is present as an axial ligand in the ferrous heme– saturated due to the high protein concentration of the crystals

nitrosyl complex of SfmD. In the presence of 3-Me-^L-Tyr, the EPR (Fig. 6A). The near fully reduced crystal (red trace) did not

spectrum showed a noticeable diﬀerence to that of the EPR diﬀract. However, we were able to obtain diﬀraction data from

spectrum of the ferrous–nitrosyl complex in the presence of 3- partially reduced single crystals. Two datasets corresponding to

Me-^L-Tyr (Fig. 5B), suggesting that substrate-binding has per- the blue and green traces shown in Fig. 6A, which still have

turbed the electronic structure of the heme center. These data noticeable shoulders from the resting state, were rened to 2.95

˚

indicate that cNO either binds to a ve-coordinate ferrous center and 3.15 A resolutions, respectively (Fig. 6B–D). The crystal

or displaces a weak histidine ligand. A mutagenesis analysis was structure corresponding to the green spectrum has 100%

also conducted with the desire to probe which of the histidine completeness (Table 1) and shown in Fig. 6B. In the green

ligands dissociates during substrate binding or chemical spectrum, the peak height at 554 nm is taller than that of

reduction. His274 and His313 were each mutated to alanine, 525 nm, and the diﬀerence between the peak intensity is

asparagine, and glutamine in separate experiments on the smaller than that of the red trace. However, no electron density

codon-optimized version of SfmD using primers listed (Table was found for 3-Me-^L-Tyr. In the crystals soaked with dithionite

S3†). However, the mutagenesis at these positions led to poor without 3-Me-^L-Tyr, similar single-crystal optical shis were

protein expression.

observed, but the corresponding crystals did not diﬀract.

The alignment of the partially reduced structure with the

initial ferric heme structure showed a global change of the

˚

Identifying the labile axial heme ligand through redox-linked

structural determinations

overall structure with an rmsd value of 0.6 A for 303 C_acarbons.

˚

The heme position shied 0.5 A from the resting state, and

His274 swings away from the iron ion with the distance between

The heme cofactor must open up a coordination position for

H₂O₂(ferric state) or O₂(ferrous state) to bind during catalysis.

Despite extensive eﬀorts, all attempts to crystallize reduced or

substrate-bound SfmD were unsuccessful, so chemical reduc-

tion of SfmD crystals was pursued. Using single-crystal UV-vis

spectroscopy, we monitored the chemical reduction of the

ferric heme in the single crystal of SfmD before and aer

soaking with dithionite in the presence of 3-Me-^L-Tyr prior to X-

ray exposure. Upon chemical reduction, the a/b region became

more pronounced at 525 and 554 nm, while the Soret band was

˚

N₃₂of His274 and heme iron increasing from 2.2 A to 2.7 A.

Moreover, a nearby distal loop containing Ser267-Gly268-

Leu269-His270-Trp271 rotates substantially and creates

a distal pocket not observed in the initial resting state (Fig. 6C

and D). These structural changes are likely preparations for

substrate binding. The side chain of Trp271, which is adjacent

to His274, ips, and undergoes a signicant conformational

change. Ser267 and Gly268 move away from the active site.

Leu269 and His270 move closer to the heme center. The main

Fig. 6 Partially reduced crystal structure of SfmD identiﬁes iron ligand His274 rotating out during active site reorganization. (A) Single-crystal UV-

vis spectroscopy of SfmD (black trace) after in crystallo reduction by dithionite in the presence of 5 mM 3-Me-^L-Tyr (blue and green traces for

partially reduced crystals, and red trace for the reduced crystal that did not diﬀract), and (B) a partially reduced structure determined from the

crystal that yielded the green spectrum in (A) and aligned against the SfmD structure in the resting state (with carbon atoms and main chains in

gray color). The carbon atoms and the main chain of the partially reduced structure are shown in green with heme in dark red. Electron density

maps for the conformational change in the partially reduced structures are shown for (C) 6VDZ and (D) 6VE0. 2F_oꢁ F_celectron density maps

from the ﬁnal structural reﬁnement are presented in blue contoured at 1s. Omit F_oꢁ F_celectron density maps for the heme ligand His274 and the

loop region containing (267–271, green) after simulated annealing are shown in orange contoured at 3s. Electron density maps are shown in two

diﬀerent orientations. A movie clip showing more detailed structural rearrangements is available in the ESI.†

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chain carbonyl oxygen of Leu269 forms a direct interaction with serine substituted variants (C317A and C317S) were generated

His274 in the partially reduced structure. Moreover, the heme based on the codon-optimized DNA sequence. C317S and

propionate groups also showed signicant rotation. A video clip C317A showed signicantly decreased heme occupancy of 4.3%

showing the structural rearrangements is available as part of and 2.7%, compared to wild-type (Fig. S15†). An equal amount

the online version of the ESI.† Overall, the partially reduced of heme content was present for both the mutants and the wild-

structure provides clues about the active site reorganization in type enzyme to oﬀset the low heme occupancy in the catalytic

response to reduction. It is worth noting that all these structural activity assay. However, the two mutants did not exhibit any

changes observed from the partially reduced enzyme were hydroxylation activity on 3-Me-^L-Tyr (Fig. S16†). Thus, the loss of

conned in a densely packed crystal lattice, and further changes catalytic activity was not caused by low heme occupancy. Addi-

would be expected in solution.

tion of heme increased heme occupancy to 40.0% and 39.9% for

C317S and C317A, respectively (Fig. S15†). However, both heme

reconstituted mutant enzymes failed to produce product

(Fig. S17†). These results suggested that Cys317 is critical for

both heme occupancy and activity. Although the variants were

catalytically inactive, we found that ascorbate was able to reduce

The single thioether covalency of the cofactor is essential for

catalytic activity and heme occupancy

To investigate the eﬀect of the single thioether covalency, we

performed a mutagenesis study on Cys317. The alanine and

Fig. 7 Mutation of Cys317 abolishes the covalency attachment of the heme and catalytic activity. (A) UV-vis spectra of SfmD C317S (black trace)

and C317S SfmD with ascorbate (blue trace); (B) the same as (A) except with the presence of 3-Me-^L-Tyr; (C) UV-vis spectra of C317S with

ascorbate (black trace) and C317S with ascorbate and cNO (blue trace); (D) the same as (C) except with the presence of 3-Me-L-Tyr. The plots for

changes at 425 nm as a function of time are shown in the inset in panels (A)–(D); (E) EPR spectra of C317S (black trace), C317S after addition of 3-

Me-L-Tyr (red trace), and C317S after addition of ascorbate (blue trace); and (F) EPR spectra of ferric C317S (gray trace), after addition of ascorbate

and cNO (black), or after addition of 3-Me-^L-Tyr, ascorbate, and cNO (blue).

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the non-covalently bound heme in C317S. However, the reduc- and monooxygenation, and other types of oxidation reactions. A

tion rate was decreased by approximately 5-fold compared to previous study of MarE suggested that it might be a TDO

the wild-type enzyme (Fig. 7A and Table S1†). A noticeable homolog despite being a monooxygenase.¹⁹Recently, incorpo-

diﬀerence from the wild-type protein is that the addition of 3- rating a single atom of oxygen into the substrate during O₂-

Me-^L-Tyr to C317S under anaerobic conditions did not cause the driven turnover was observed for both IDO and TDO on their

blue shi of the Soret at 411 nm (Fig. 7C). Both the ligand-free native substrates²¹or a tryptophan analog in which the

and substrate-bound C317S proteins showed a similar red- substrate amino group is replaced by a hydroxyl group.²³The

shied Soret reduction pattern. Next, cNO was introduced monooxygenase activity of TDO on its native substrate can also

under the O₂-free conditions to C317S which was reduced by be achieved as the primary reaction through directed evolu-

ascorbate in the absence and presence of 3-Me-L-Tyr (Fig. 7C tion.³⁹Hence, the core structural platform within the His-

and D). The addition of cNO showed a similar blue-shi pattern ligated, heme-dependent oxygenase superfamily is suitable for

observed in wild-type enzymes with a relatively faster rate. In various oxygenation reactions. The nding of the common

sharp contrast to the wild-type protein by which the addition of structural platform may facilitate future mechanistic investi-

substrate 3-Me-^L-Tyr converted the near EPR-invisible HALS gations of the newly annotated members.

heme to a high-spin EPR-active state, the addition of substrate

SfmD shows a redox-linked metal–ligand dissociation upon

did not cause an observable change to the ferric form of the reduction and a non-redox-linked ligand displacement upon

mutant (Fig. 7E). Next, cNO was introduced to the ascorbate substrate binding. The heme iron is coordinated by two histi-

reduced samples (Fig. 7F). Unlike the wild-type protein, the EPR dine residues with no available coordination spot to bind

spectra of the ferrous heme–nitrosyl complex of C317S in the external molecules in the resting state, making a recalcitrant

absence and presence of the substrate showed identical EPR active site for the enzyme. One plausible reason for the six-

spectra. The EPR spectra of these ferrous heme nitrosyl coordination is that SfmD is not in a metabolic pathway for

complexes are distinct from the corresponding data of wild-type energy extraction or essential for cell division. Instead, the

enzyme. Instead, the spectra resemble that of a closely related biosynthetic pathway containing SfmD is used for producing

enzyme, i.e., the b-type heme-containing tyrosine hydroxylase, unique molecules such as antibiotics. High activity of a non-

LmbB2.¹⁰Collectively, these results indicated that the loss of the essential pathway may unnecessarily reduce the cellular avail-

single cysteine–vinyl covalent linkage through mutation led to ability of tyrosine. Moreover, SfmD is capable of hydroxylating ^L-

heme with an electronic structure reminiscent of b-type heme Tyr directly. Although ^L-Tyr is a less eﬃcient substrate, it is

and results in a catalytically inactive variant.

present in cells at a much higher concentration than 3-Me-^L-Tyr.

Therefore, the locked heme center in SfmD is likely a regulatory

safety control to prevent ^L-Tyr depletion or uncoupled oxygen

activation. Dissociation of His274 from the heme iron with

Discussion

Oxidative, mononuclear heme-based enzymes include cysteine- accompanying loop movements to reconstruct the active site

ligated superfamilies (cytochrome P450 and peroxygenase) and only occurs upon chemical reduction of the heme cofactor or

histidine-ligated superfamilies (peroxidase and dioxygenase). substrate binding to the distal pocket when the intended reac-

Our recent work on TDO and related heme-dependent tion is ready to proceed. Redox-linked changes in ligand coor-

enzymes,^{10,23,33–38}especially tyrosine hydroxylase (TyrH), dination or dissociation, resulting in protein conformational

prompted us to explore the structural and functional relation- rearrangements, are more typically observed in sensor proteins

ships of these enzymes, focusing on the question of whether or and engineered cytochrome c proteins.⁴⁰

not a group of histidine-ligated heme-based enzymes can acti-

Ligand dissociation due to heme reduction to the ferrous

vate oxygen for monooxygenation and how this group may oxidation state is also not uncommon, and such a phenomenon

relate to other categories of mononuclear heme-based enzymes. has been described in several systems. Redox-linked ligand

In this work, integrated biochemical, spectroscopic, and struc- switching and/or dissociation has been commonly found in

tural studies have shown that (1) SfmD is a monooxygenase bacterial periplasmic diheme cytochrome protein, such as

which can mediate an ascorbate/O₂-dependent catalytic mono- cytochrome c peroxidase (bCcP),⁴¹thiosulfate dehydrogenase,⁴²

oxygenation of its native substrate, (2) the de novo SfmD crystal and cytochrome cd₁, nitrite reductase.⁴³Upon reduction, an

structure and a partially reduced crystal structure reveal an endogenous heme ligand dissociation occurs, thereby opening

unprecedented Hx_nHxxxC (n ꢀ 38) heme-binding motif of single up one axial position for direct heme iron ligation of an external

thioether link and a bis-His ligand set where the rst histidine substrate such as H₂O₂, nitrite, or thiosulfate. Another redox-

in the motif dissociates during catalysis, while the second regulated ligand switching phenomenon has been reported in

histidine and cysteine residues (the HxxxC portion of the ligand b-type heme-dependent 15-cis-z-carotene isomerase.⁴⁴In a non-

set) remain bound to the prosthetic group, and (3) SfmD is catalytic system, heme–ligand switching has been observed,

shown to be a structurally validated prototypic monooxygenase though usually with a weak heme–thiolate bond and a heme

in the superfamily currently known as the heme-dependent dissociation rate constant that is 10⁷larger than His-ligated.^45,46

dioxygenase superfamily.

The labile nature of the Fe–S bond is fundamental to some

Functionally, the members of the His-ligated, heme- heme–thiolate complexes with six-coordinate and low-spin

dependent oxygenase superfamily can utilize O₂as the elec- heme as a heme sensor or transporter. In cytochrome c, one

tron acceptor for a variety of reactions, including dioxygenation of the heme ligands, Met80, is replaced with a lysine residue

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upon transition to an alkaline environment. The loss of Met80 constructed expression vector, although pET-37b has a signal

is essential for turning cytochrome c into a peroxidase that is sequence on its backbone. In both studies, the Ccm system was

required for programmed cell death.^47,48

not co-transformed during protein production in E. coli BL21

To the best of our knowledge, an endogenous metal–ligand (DE3).

dissociation in response to the primary organic substrate in

Autocatalytic c-type heme maturation is, however, not a new

ferric heme enzymes is a less common mechanism. A similar phenomenon. It has been observed in the expression of

scenario has been previously described by Lipscomb and Hydrogenobacter thermophilus cytochrome c₅₅₂in E. coli without

colleagues in a non-heme iron-dependent intradiol dioxygenase a periplasmic leader sequence,⁵⁹and in E. coli Ccm deleted

enzyme, i.e., protocatechuate 3,4-dioxygenase (3,4-PCD), where strain.⁶⁰The c-type thioether bonds in cytochrome c₅₅₂can be

a metal–ligand, Tyr447, is displaced by the substrate during formed between heme and apoprotein in vitro in the absence of

catalysis.^49,50Another example has been described in the crystal any maturation system.⁶¹Likewise, cysteine-based autocatalytic

structures of dehaloperoxidase from Amphitrite ornate in crosslinking involving a protein cysteine residue has been

complex with various guaiacol substrates.⁵¹In the crystal described in other copper- and iron-dependent proteins.^62–65

structures of dehaloperoxidase, one subunit is substrate-free Similarly, an autocatalytic, covalent methionine–heme linkage

containing a bis-His ligated b-type heme, while the other has been observed in ascorbate peroxidase.⁶⁶A mechanism for

subunit is substrate-bound and with one of the histidine the autocatalytic crosslink has been proposed via electrophilic

ligands dissociated from the heme.

addition. Increased electron density in the heme would aﬀord

In the newly discovered Hx_nHxxxC heme-binding motif (n ꢀ the protonation on the heme vinyl group, followed by the

38), the HxxxC fragment is the most critical portion. Neither the formation of carbocation for the electrophilic addition of the

entire motif nor the more catalytically critical HxxxC ligand set crosslinking residue.⁶⁷The autocatalytic crosslinking mecha-

has previously been linked to a heme prosthetic group in nism in SfmD is not in the scope of this work and remains to be

enzymes. The HxxxC motif has been found, or proposed, in investigated in future studies.

a few proteins for binding iron–sulfur cluster or zinc ion.^52–55

A

The eﬀects of heme crosslinking to a protein, other than

similar, CxxHx₁₆H heme-binding motif has been reported in the securing the heme, have been studied in other metalloproteins.

K_ATPchannel protein, whose ion channel opens in response to Hydroxylamine oxidizing cytochrome P460 has a unique lysine

the binding of carbon monoxide.⁵⁶A signicant diﬀerence is crosslinked c-type monoheme.⁶⁸This heme–Lys crosslink plays

that the cysteine residue in this motif is an axial ligand of the a role as a mechanical restraint to bring the secondary sphere

heme cofactor which does not form a covalent link to a vinyl glutamate residue close enough to promote catalysis. A hemo-

group of the heme, as observed in this study of SfmD.

globin, GlbN-A from cyanobacterium Synechococcus, is capable

The inspection of the crystal structure of SfmD showed of showing cNO dioxygenase activity and has an unusual heme–

noticeable diﬀerences from the structure of bacterial TDO.⁵⁷His crosslink installed by protein posttranslational modica-

The heme groups of the two proteins are in adjacent cles of tion.⁶⁹Preimesberger and colleagues investigated the eﬀect of

their respective enzymes, and they are anked by Trp253 and the heme–His crosslink on the reactivity of cNO. Both forms of

Trp271 on opposite faces, proximal for TDO and distal for SfmD the protein with (GlbN-A) and without His–heme crosslink

(Fig. 4E). His274, in the previously proposed SGxxxxDH motif,²(GlbN) show cNO oxidation activity, but the His–heme (GlbN-A)

turned out to be one of the axial His ligands. Nevertheless, in abolished cNO reduction chemistry to HNO. The diﬀerent

the partially reduced structures of SfmD, the loop region in this reactivity suggests the covalent modication of the heme plays

SGxxxxDH motif shows signicant rearrangement (Fig. 6B–D), a role as a chemistry modulator.⁶⁹

suggesting a possible functional relevance.

Compared to the crystal structure of the resting state, the

The vast majority of canonical c-type heme attachments in E. partially reduced crystal structures have severely reduced reso-

coli expressed proteins mature with the aid of the cytochrome c lution and higher B-factors. Upon completion of the chemical

maturation (Ccm) system in the periplasm.⁵⁸However, in the reduction, the SfmD single crystals no longer diﬀract X-rays,

preparation of SfmD protein expressed in E. coli, the covalency suggesting that the crystal packing has been perturbed. The

through Cys317 in HxxxC motif is achieved without co- partially reduced crystal structures were captured before the

transformation of the Ccm system. Analysis of the primary crystals completely lose their diﬀractive structural integrity.

sequence of SfmD with SingalP (see ESI,† Materials and Therefore, overall crystal packing was aﬀected by substantial

methods) indicated that there is no cleavable periplasmic conformational changes, thereby losing its high-resolution

secretion signal peptide present. It is worth mentioning that diﬀraction with an accompanying increase in B-factors. Unfor-

neither our N-terminal cleavable His-tagged version of SfmD tunately, the current crystal lattice only allows modest changes,

used in this study nor the C-terminal non-cleavable His-tagged which cannot accommodate substrate binding while still dif-

˚

version in the previous study have a periplasmic localization fracting to around 3 A resolution. Despite the decrease in

leader sequence. Yet, the resulting proteins are catalytically resolution, the F_oꢁ F_comit electron density maps aer simu-

active towards the native substrate. The construction of the C- lated annealing show signicant rearrangement of the loop

terminal non-cleavable His-tagged version in the pET-37b region near the heme, suggesting that the distal ferrous heme

expression vector in the previous study was achieved by NdeI pocket is signicantly diﬀerent than the ferric heme in this

and HindIII;²thus, it is not possible to have a leader sequence region.

between the ribosome binding site and SfmD sequence in the

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The basis of SfmD categorization in the oxygen utilizing monooxygenase members. Its structural resemblance to the

tryptophan dioxygenase superfamily might be questioned due heme-dependent dioxygenase members in the TDO superfamily

to its ability to utilize H₂O₂to carry out oxygenation. However, presents the implication of mechanistic understandings among

preoxygenation activity has been reported for IDO when H₂O₂is this group of enzymes.

used as the oxidant.⁷⁰Another well-informed monooxygenase

superfamily with a cysteine-ligated heme, cytochromes P450,

can also utilize both dioxygen and hydrogen peroxide. The

peroxide pathway is known as a catalytic shunt or the short

Experiment procedures

Materials and methods

circuit in the monooxygenase reactions.^29,71,72Hence, a similar The detailed methods and materials are provided in ESI,†

shunt pathway may be proposed for SfmD and analogs. We have including the synthesis and characterization of 3-Me-^L-Tyr,

previously found that H₂O₂can reactivate ferric TDO in the cloning, expression, and purication of SfmD, crystallization,

presence of ^L-tryptophan via a ferryl intermediate.³³The loca- data collection, and structure determination, description of

tion and surroundings of the active site and availability of enzyme reaction experiments, HPLC, mass spectrometry, UV-

oxidants are likely determining factors for selecting oxidants. vis, and electron paramagnetic resonance (EPR) spectroscopy,

Given the ability of ascorbate to reduce the heme–iron of SfmD, characterization of the enzyme and the reaction products,

it may be expected to be in its ferrous, oxygen-utilizing state in sequence and structural alignments.

a typical cellular context.

As a physiologically relevant reducing agent, ascorbate

Data availability

reduces ferrylmyoglobin (Fe⁴⁺]O) and metmyoglobin (Fe³⁺),

and oxidizes oxymyoglobin (Fe²⁺–O₂).⁷³Exposure of myoglobin All data are included in this manuscript and ESI,† including

to H₂O₂leads to the formation of heme–myoglobin crosslink a video. The X-ray structures of SfmD were deposited in the

accompanying the increase of peroxidase activity and the Protein Data Bank under accession codes 6VDP, 6VDQ, 6VDZ,

formation of higher oligomeric states. Ascorbate reverses such and 6VE0.

oxidative modications.⁷⁴Ascorbate can facilitate the heme

oxygenase reaction in place of NADPH/cytochrome P450

Conﬂicts of interest

reductase.⁷⁵The determined midpoint reduction potential of

SfmD is an apparent redox potential, E^a_m^ppbecause this is not the The authors declare no competing nancial interest.

case of ve-coordinate Fe(^III)–heme to ve-coordinate Fe(II)–

heme, or six-coordinate to six-coordinate, instead it is six-

coordinate Fe(^III) to ve-coordinate Fe(II) with associated heme

Acknowledgements

and protein conformational changes. Upon discovering that This work was nancially supported by the National Institutes

ascorbate behaves as a cosubstrate for SfmD in this study and of Health (NIH) grant GM108988 (to A. L.). A. L. acknowledges

MarE in a previous study,¹⁹the question naturally arises as to the Lutcher Brown Endowment support. The Center for Inno-

whether ascorbate is present in Streptomyces. The hallmark vative Drug Discovery is funded by the Cancer Research

gene responsible for ascorbate biosynthesis is ^L-gulonolactone Prevention & Research Institute of Texas (CPRIT) core facility

oxidase (Gulo), which is impaired as a pseudogene in humans.⁷⁶award RP160844 (to S. M.). We are in debt to Dr Gongli Tang for

In a protein blast search with Gulo from Rattus norvegicus, a generous gi of the non-cleavable His-tagged version of SfmD

a gene with the accession number of WP_030235215 is found in plasmid used in this study for the initial and comparative

Streptomyces lavendulae showing 35% sequence identity with studies. We thank Dr Wendell P. Griﬃth for his assistance in

98% coverage. In some microorganisms, ^D-erythroascorbate HRMS analysis. We are grateful to the staﬀ scientists at the

replaces the role of ascorbate. In this case, ^D-arabinono 1,4- beamline 9–2 of the Stanford Synchrotron Radiation Light-

lactone oxidase (ALO1) catalyzes the same chemistry as Gulo.⁷⁷source (SSRL), SLAC National Accelerator Laboratory and the

Blast search with yeast ALO1 fetches the same gene sections 19 beamlines of the Advanced Photon Source (APS),

WP_030235215, which shares a 25% sequence identity with Argonne National Laboratory for assistance in the remote

yeast ALO1. The presence of WP_030235215 lends support to single-crystal spectroscopy (SSRL only) and subsequent X-ray

the availability of ascorbate in Streptomyces lavendulae. data collections (APS and SSRL). Use of the APS and SSRL is

However, the actual electron and proton donor in the cellular supported by the U.S. Department of Energy, Oﬃce of Science,

context remains to be studied.

Oﬃce of Basic Energy Sciences, under Contracts DE-AC02-

In summary, a novel heme cofactor with a single thioether 06CH11357 and DE-AC02-76SF00515, respectively. The SSRL

covalent link and a bis-His axial ligand set discovered in this facility is additionally supported by the National Institutes of

work by spectroscopic and structural studies shows how an Health Grant P41GM103393.

enzyme holds its heme prosthetic group tightly while allowing

more exible substrate-triggered structural changes. The heme

center is recalcitrant to react with alternative substrates present

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Article Doi

A novel catalytic heme cofactor in SfmD with a single thioether bond and abis-His ligand set revealed by ade novocrystal structural and spectroscopic study

DOI: 10.1039/d0sc06369j

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Full text of DOI:10.1039/d0sc06369j

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