Ci a ion: Rixen, S.; Indo , P.M.;
Kubi za, C.; S uwe, M.A.; Klopp, C.;
Scheidig, A.J.; Kunze, T.; Clemen , B.
Reduc ion o Hyd ogen Pe oxide by
Human Mi ochond ial Amidoxime
Reducing Componen Enzymes.
Molecules 2023,28, 6384. h ps://
doi.o g/10.3390/molecules28176384
Academic Edi o : Domenico Osella
Recei ed: 1 Augus 2023
Re ised: 23 Augus 2023
Accep ed: 29 Augus 2023
Published: 31 Augus 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
molecules
A icle
Reduc ion o Hyd ogen Pe oxide by Human Mi ochond ial
Amidoxime Reducing Componen Enzymes
Sophia Rixen 1,† , Pa ick M. Indo 1,†, Ch is ian Kubi za 2, Michel A. S uwe 1,2 , Ca h in Klopp 1,2 ,
Axel J. Scheidig 2, Thomas Kunze 1and Be nd Clemen 1,*
1Depa men o Pha maceu ical and Medicinal Chemis y, Pha maceu ical Ins i u e, Kiel Uni e si y,
24118 Kiel, Ge many; [email p o ec ed] (S.R.); [email p o ec ed] (P.M.I.);
[email p o ec ed] (M.A.S.); cklopp@s ubio.uni-kiel.de (C.K.);
[email p o ec ed] (T.K.)
2Depa men o S uc u al Biology, Zoological Ins i u e, Kiel Uni e si y, 24118 Kiel, Ge many;
[email p o ec ed] (C.K.); [email p o ec ed] (A.J.S.)
*Co espondence: [email p o ec ed]
†These au ho s con ibu ed equally o his wo k.
Abs ac :
The mi ochond ial amidoxime educing componen (mARC) is a human molybdoenzyme
known o ca alyze he educ ion o a ious N-oxygena ed subs a es. The physiological unc ion o
mARC enzymes, howe e , emains unknown. In his s udy, we examine he educ ion o hyd ogen
pe oxide (H
2
O
2
) by he human mARC1 and mARC2 enzymes. Fu he mo e, we demons a e an
inc eased sensi i i y owa d H
2
O
2
o HEK-293T cells wi h an MTARC1 knockou , which implies
a ole o mARC enzymes in he cellula esponse o oxida i e s ess. H
2
O
2
is a eac i e oxygen
species (ROS) o med in all li ing cells in ol ed in many physiological p ocesses. Fu he mo e, H
2
O
2
cons i u es he i s mARC subs a e wi hou a ni ogen–oxygen bond, implying ha mARC enzymes
may ha e a subs a e spec um going beyond he p e iously examined N-oxygena ed compounds.
Keywo ds: eac i e oxygen species; hyd ogen pe oxide; molybdenum enzyme
1. In oduc ion
The human mARC enzyme was i s desc ibed in 2006 as he hi d componen o he
N- educing complex oge he wi h hemop o ein cy och ome b5 (Cyb5B) and la op o ein
cy och ome b5 educ ase (Cyb5R3) [
1
]. Toge he wi h hese wo elec on ca ie p o eins,
mARC enzymes educe a ious N-hyd oxyla ed subs a es like amidoximes, N-hyd oxy-
guanidines, hyd oxylamines, N-oxides o hyd oxamic acids [
2
]. mARC u ilizes a Mo-
molybdop e in co ac o (Moco), he coo dina ion o he ca aly ic molybdenum ion being
e y simila o ha obse ed in sul i e oxidase (SO) [
3
,
4
] despi e no sha ing many o he
cha ac e is ics wi h SO. Thus, mARC enzymes a e classi ied as pa o a sepa a e, new amily
o molybdenum enzymes, he MOSC domain amily [
5
,
6
]. All mammalian genomes encode
wo pa alogues o mARC: mARC1 and mARC2 (gene names MTARC1,MTARC2) [4].
Hyd ogen pe oxide (H
2
O
2
) is he majo eac i e oxygen species (ROS) in euka yo ic
cells and some 37 human enzymes a e known o gene a e H
2
O
2
by he wo-elec on
educ ion o dioxygen [
7
]. In e es ingly, among hese H
2
O
2
-p oducing enzymes a e he
euka yo ic molybdenum enzymes xan hine oxidase (XO) [
8
], aldehyde oxidase (AO) [
9
]
and sul i e oxidase (SO) [10].
While high concen a ions o H
2
O
2
cause oxida i e damage o cells, i is conside ed o
be a physiologically ele an signaling molecule a lowe concen a ions. These di e en
e ec s o H2O2ha e been e iewed in de ail elsewhe e [7].
Va ious cell compa men s con ain H
2
O
2
-deg ading enzymes, ensu ing igh egu-
la ion o H
2
O
2
concen a ions. Examples a e ca alase o myelope oxidase, which, due
o hei high K
M
alues, a e sui able o deg ading high H
2
O
2
concen a ions, e.g., in
Molecules 2023,28, 6384. h ps://doi.o g/10.3390/molecules28176384 h ps://www.mdpi.com/jou nal/molecules
Molecules 2023,28, 6384 2 o 12
pe oxisomes [11,12]. O he enzymes like GPx1 o pe oxi edoxins ac a much lowe H2O2
concen a ions cha ac e is ic o hei espec i e cell compa men s [13,14].
Un il now, no H
2
O
2
-deg ading enzyme has been iden i ied in he ou e mi ochond ial
memb ane (OMM), whe e mARC enzymes a e localized [15].
The mARC enzyme sys em is known bes o i s educ i e ac i i y owa d N-oxygena ed
compounds. Howe e , some s udies ha e shown links be ween mARC and ROS. Fo ex-
ample, he common mARC1 p.A165T a ian is associa ed wi h highe le els o lipid
pe oxida ion, while he o al an ioxidan ac i i y (TAA) in se um and exp ession o ca alase
a e inc eased [16].
In his wo k, we p esen he NADH-dependen deg ada ion o H
2
O
2
by he human
mARC1 and mARC2 enzymes in conce wi h hei elec on ca ie s Cyb5B and CYB5R3
using ecombinan p o eins. We go on o show he e ec o an MTARC1 knockou on he
iabili y o HEK-293T cells in he p esence o high ex e nal H2O2concen a ions.
2. Resul s
2.1. Molybdenum-Con aining mARC1 and mARC2 Bo h Reduce H2O2
To assess he educ ion o H
2
O
2
by mARC enzymes, we compa ed he NADH con-
sump ion, measu ed in he NADH assay, and he amoun o esidual H
2
O
2
, quan i ied by
he luo ome ic assay, o se e al di e en se ups. Impo an ly, ex ensi e con ol eac ions
we e examined o unambiguously iden i y he e ec o molybdenum-con aining mARC
enzymes. The esul s om hese assays a e isualized in Figu e 1. No e ha di e en H
2
O
2
concen a ions (50
µ
M o mARC1 and 80
µ
M o mARC2) we e used due o di e en
s abili ies o he enzymes owa d high H2O2concen a ions.
Molecules2023,28,xFORPEERREVIEW2o 12


hei highK
M
 alues,a esui able o deg adinghighH
2
O
2
concen a ions,e.g.,inpe oxi-
somes[11,12].O he enzymeslikeGPx1o pe oxi edoxinsac a muchlowe H
2
O
2
con-
cen a ionscha ac e is ic o  hei  espec i ecellcompa men s[13,14].
Un ilnow,noH
2
O
2
-deg adingenzymehasbeeniden i iedin heou e mi ochond ial
memb ane(OMM),whe emARCenzymesa elocalized[15].
ThemARCenzymesys emisknownbes  o i s educ i eac i i y owa dN-oxygen-
a edcompounds.Howe e ,somes udiesha eshownlinksbe weenmARCandROS.Fo 
example, hecommonmARC1p.A165T a ian isassocia edwi hhighe le elso lipid
pe oxida ion,while he o alan ioxidan ac i i y(TAA)inse umandexp essiono ca a-
lasea einc eased[16].
In hiswo k,wep esen  heNADH-dependen deg ada iono H
2
O
2
by hehuman
mARC1andmARC2enzymesinconce wi h hei elec onca ie sCyb5BandCYB5R3
using ecombinan p o eins.Wegoon oshow heeffec o anMTARC1knockou on he
iabili yo HEK-293Tcellsin hep esenceo highex e nalH
2
O
2
concen a ions.
2.Resul s
2.1.Molybdenum-Con ainingmARC1andmARC2Bo hReduceH
2
O
2

Toassess he educ iono H
2
O
2
bymARCenzymes,wecompa ed heNADHcon-
sump ion,measu edin heNADHassay,and heamoun o  esidualH
2
O
2
,quan i iedby
he luo ome icassay, o se e aldiffe en se ups.Impo an ly,ex ensi econ ol eac-
ionswe eexamined ounambiguouslyiden i y heeffec o molybdenum-con aining
mARCenzymes.The esul s om heseassaysa e isualizedinFigu e1.No e ha di -
e en H
2
O
2
concen a ions(50µM o mARC1and80µM o mARC2)we euseddue o
diffe en s abili ieso  heenzymes owa dhighH
2
O
2
concen a ions.

Figu e1.S ackedba -cha  ep esen ing heconsump iono NADH(b owncolumns)on opo  he
esidualamoun o H
2
O
2
(bluecolumns) o mARC1(panelA)andmARC2(panelB).Theindi idual
se upscompa ed oeacho he a e( omle  o igh ):nop o ein—assaycon ainingonlyNADH
andH
2
O
2
bu noenzymes;mARC1/2comple e—con ainsmARC1o mARC2andbo helec onca -
ie p o eins;apo-mARC1comple e—sameasbe o ebu wi hmolybdenum- eeapo-mARC;only
mARC1/2—jus mARC1/2,bu noelec onca ie s;onlyCyb5BandCyb5R—onlyelec onca ie s
bu nomARCenzymes.
I isclea lyobse ed ha , o bo hmARC1andmARC2, heby a g ea es deple ion
o NADHisobse edwhen hecomple e, econs i u edmARC1/2enzymesys emsa e
used.Co espondingly,in hese eac ions, helowes concen a ionso  esidualH
2
O
2
we e
ound,whichcon i ms ha NADHconsumedby hemARCenzymesys emdoesin ac 
educeH
2
O
2
.Thecon ol eac ionsindica e ha onlyholo-mARCenzymeswi hamolyb-
dop e inp os he icg oupcan educeH
2
O
2
inconce wi hCyb5BandCyb5R3.
Figu e 1.
S acked ba -cha ep esen ing he consump ion o NADH (b own columns) on op o he
esidual amoun o H
2
O
2
(blue columns) o mARC1 (panel A) and mARC2 (panel B). The indi idual
se ups compa ed o each o he a e ( om le o igh ): no p o ein—assay con aining only NADH
and H
2
O
2
bu no enzymes; mARC1/2 comple e—con ains mARC1 o mARC2 and bo h elec on
ca ie p o eins; apo-mARC1 comple e—same as be o e bu wi h molybdenum- ee apo-mARC; only
mARC1/2—jus mARC1/2, bu no elec on ca ie s; only Cyb5B and Cyb5R—only elec on ca ie s
bu no mARC enzymes.
I is clea ly obse ed ha , o bo h mARC1 and mARC2, he by a g ea es deple ion
o NADH is obse ed when he comple e, econs i u ed mARC1/2 enzyme sys ems a e
used. Co espondingly, in hese eac ions, he lowes concen a ions o esidual H
2
O
2
we e ound, which con i ms ha NADH consumed by he mARC enzyme sys em does
Molecules 2023,28, 6384 3 o 12
in ac educe H
2
O
2
. The con ol eac ions indica e ha only holo-mARC enzymes wi h a
molybdop e in p os he ic g oup can educe H2O2in conce wi h Cyb5B and Cyb5R3.
2.2. Kine ics o mARC-Dependen H2O2Reduc ion
Bo h mARC1 and mARC2 display Michaelis–Men en kine ics o H
2
O
2
educ ion, as is
shown in Figu e 2. The u no e a es and K
M
alues o H
2
O
2
educ ion by mARC1
and mARC2 a e compa able, wi h mARC1 showing a sligh ly lowe K
M
bu highe
u no e a es.
Molecules2023,28,xFORPEERREVIEW3o 12
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
2.2.Kine icso mARC-Dependen H
2
O
2
Reduc ion
Bo hmARC1andmARC2displayMichaelis–Men enkine ics o H
2
O
2
 educ ion,as
isshowninFigu e2.The u no e  a esandK
M
 alues o H
2
O
2
 educ ionbymARC1and
mARC2a ecompa able,wi hmARC1showingasligh lylowe K
M
bu highe  u no e 
a es.

Figu e2.(A)Kine icp o ileo mARC1-andmARC2-ca alyzedH
2
O
2
 educ ion.(B)Kine icpa am-
e e sob ained om i ing o heMichaelis–Men enequa ion.Valuesinpa en hesesindica e he
95%likelihoodin e al o K
M
andV
max
.
Usingou  ecen lyes ablished luo escence-basedhigh- h oughpu assay[17],we
we eable omeasu e e ysimila con e sion a es o  hemARC-ca alyzed educ iono 
hyd ogenpe oxide.
2.3.MTARC1Knockou Dec easesCellViabili yinP esenceo H
2
O
2
Tode e minewhe he o no  he educ iono H
2
O
2
by ecombinan mARCenzymes
is ele an incellulo,weexamined heimpac o diffe en H
2
O
2
concen a ionsoncell i-
abili yusinganHEK-293T-basedknockou model.SinceHEK-293Tcellsexp essonly e y
lowle elso mARC2, heMTARC1knockou  esul sincellsp ac icallyde oido mARC
ac i i y(mARC2exp essionle elsdono inc ease ocompensa e heMTARC1knockou ,
.Theknockou wasshown obeeffec i eon hep o einle elbyWes e nblo analysis
(Figu e3).Be o eincuba ionwi hH
2
O
2
,somecellswe e ea edwi hbu hioninesul-
oximine(BSO),aninhibi o o glu a hionesyn hesis.
Figu e 2.
(
A
) Kine ic p o ile o mARC1- and mARC2-ca alyzed H
2
O
2
educ ion. (
B
) Kine ic pa ame-
e s ob ained om i ing o he Michaelis–Men en equa ion. Values in pa en heses indica e he 95%
likelihood in e al o KMand Vmax.
Using ou ecen ly es ablished luo escence-based high- h oughpu assay [
17
], we
we e able o measu e e y simila con e sion a es o he mARC-ca alyzed educ ion o
hyd ogen pe oxide.
2.3. MTARC1 Knockou Dec eases Cell Viabili y in P esence o H2O2
To de e mine whe he o no he educ ion o H
2
O
2
by ecombinan mARC enzymes
is ele an in cellulo, we examined he impac o di e en H
2
O
2
concen a ions on cell
iabili y using an HEK-293T-based knockou model. Since HEK-293T cells exp ess only
e y low le els o mARC2, he MTARC1 knockou esul s in cells p ac ically de oid o
mARC ac i i y (mARC2 exp ession le els do no inc ease o compensa e he MTARC1
Molecules 2023,28, 6384 4 o 12
knockou . The knockou was shown o be e ec i e on he p o ein le el by Wes e n blo
analysis (Figu e 3). Be o e incuba ion wi h H
2
O
2
, some cells we e ea ed wi h bu hionine
sul oximine (BSO), an inhibi o o glu a hione syn hesis.
Molecules2023,28,xFORPEERREVIEW4o 12
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

Figu e3.P o einp oduc ion o e i yMTARC1KO.MTARC1
−/−
andWTcellswe elysed,36µgo 
p o einwasappliedpe laneandWes e nblo analyseswe epe o medusinganan i-mARC1an-
ibody.
Diffe encesseenbe weenwild ypeandknockou cellsa eal eady e lec edbycell
mo phology.Achangedcellmo phologyinducedbyH
2
O
2
,whichcanbeobse edinKO
cellsa 20µM,onlyoccu sinWTcellsa 30µM,while heKOcellsa 30µMcanha dly
beconside edmo phologicallyali e(Figu e4).Fu he mo e,Hoechs s aining e ealed
ha knockou cells ea edwi h30µMH
2
O
2
hadhighe nuclea condensa ionand he eby
aninc easedapop osis a ecompa ed owild ypecells(Figu e5C).

Figu e4.Cy o oxici yo H
2
O
2
 oHEK-293TMTARC1-KOandWTcells.Cellswe eseededon o96-
wellpla esandincuba edwi hmediumcon aining0.3mMBSO o 16h, ollowedbyincuba ion
Figu e 3.
P o ein p oduc ion o e i y MTARC1 KO. MTARC1
−/−
and WT cells we e lysed,
36
µ
g o p o ein was applied pe lane and Wes e n blo analyses we e pe o med using an
an i-mARC1 an ibody.
Di e ences seen be ween wild ype and knockou cells a e al eady e lec ed by cell
mo phology. A changed cell mo phology induced by H
2
O
2
, which can be obse ed in KO
cells a 20
µ
M, only occu s in WT cells a 30
µ
M, while he KO cells a 30
µ
M can ha dly be
conside ed mo phologically ali e (Figu e 4). Fu he mo e, Hoechs s aining e ealed ha
knockou cells ea ed wi h 30 µM H2O2had highe nuclea condensa ion and he eby an
inc eased apop osis a e compa ed o wild ype cells (Figu e 5C).
This obse a ion was con i med in a esazu in-based cell iabili y assay. While low
concen a ions o H
2
O
2
do no appea o ha e a nega i e in luence on cell iabili y in
ei he WT o KO cells, when inc easing H
2
O
2
concen a ions abo e 10
µ
M, WT and KO
cells clea ly show di e gence, wi h he iabili y o WT cells being signi ican ly highe
(Figu e 5A,B). A dec eased iabili y o knockou cells can al eady be obse ed a e 8 h
and only becomes e en mo e p onounced a e longe incuba ion pe iods. A e 48 h,
KO cells a e no iable a 30
µ
M H
2
O
2
, whe eas he same is obse ed wi h WT cells a a
concen a ion o 80 µM H2O2. A a concen a ion o 30 µM, he iabili y o he WT cells is
s ill app ox. 70%.
These indings show ha H
2
O
2
deg ada ion by he mARC1 enzyme does occu
in cell cul u e, and i has a measu able e ec on cell physiology a high ex acellula
H2O2concen a ions.
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Figu e3.P o einp oduc ion o e i yMTARC1KO.MTARC1
−/−
andWTcellswe elysed,36µgo 
p o einwasappliedpe laneandWes e nblo analyseswe epe o medusinganan i-mARC1an-
ibody.
Diffe encesseenbe weenwild ypeandknockou cellsa eal eady e lec edbycell
mo phology.Achangedcellmo phologyinducedbyH
2
O
2
,whichcanbeobse edinKO
cellsa 20µM,onlyoccu sinWTcellsa 30µM,while heKOcellsa 30µMcanha dly
beconside edmo phologicallyali e(Figu e4).Fu he mo e,Hoechs s aining e ealed
ha knockou cells ea edwi h30µMH
2
O
2
hadhighe nuclea condensa ionand he eby
aninc easedapop osis a ecompa ed owild ypecells(Figu e5C).

Figu e4.Cy o oxici yo H
2
O
2
 oHEK-293TMTARC1-KOandWTcells.Cellswe eseededon o96-
wellpla esandincuba edwi hmediumcon aining0.3mMBSO o 16h, ollowedbyincuba ion
Figu e 4.
Cy o oxici y o H
2
O
2
o HEK-293T MTARC1-KO and WT cells. Cells we e seeded on o
96-well pla es and incuba ed wi h medium con aining 0.3 mM BSO o 16 h, ollowed by incuba ion
wi h medium con aining 20–80
µ
M H
2
O
2.
A e 48 h o incuba ion, cell mo phology was examined
mic oscopically. A 0–20
µ
M H
2
O
2
, bo h WT and KO cells ha e e y simila mo phologies. Howe e ,
when he H
2
O
2
concen a ion is inc eased o 30
µ
M, mo phology o KO cells changes d as ically,
whe eas he WT cells look la gely una ec ed. A 80
µ
M, bo h cell lines display s ong mo phological
di e ences, esembling he changes al eady seen a 30 µM o KO cells.
Fu he , an in luence on cell p oli e a ion could be obse ed. Ex acellula concen a-
ions o 10
µ
M H
2
O
2
showed no impai men on cell p oli e a ion. An ex acellula H
2
O
2
concen a ion o 20
µ
M did lead o impai ed cell p oli e a ion: a e 24 h, he p oli e a ion
o bo h WT and KO cells was dec eased compa ed o con ol cells wi hou H
2
O
2
ea men .
This impai men on cell p oli e a ion was mo e p onounced in mARC1-de icien cells,
whe e, a e 24 h, only 50% could be coun ed compa ed o cells ea ed wi h he medium
only; hus, in pu ely a i hme ical e ms, no cell di ision o he KO cells had aken place in
he las 24 h. The numbe o WT cells was educed o app ox. 70%. A e 72 h, bo h WT and
KO cells we e educed o app ox. 35%. A a H
2
O
2
concen a ion o 30
µ
M, no measu able
cell di ision occu ed in ei he he KO o WT cells (Figu e 6).

Molecules 2023,28, 6384 6 o 12
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wi hmediumcon aining20–80µMH
2
O
2.
A e 48ho incuba ion,cellmo phologywasexamined
mic oscopically.A 0–20µMH
2
O
2
,bo hWTandKOcellsha e e ysimila mo phologies.Howe e ,
when heH
2
O
2
concen a ionisinc eased o30µM,mo phologyo KOcellschangesd as ically,
whe eas heWTcellslookla gelyunaffec ed.A 80µM,bo hcelllinesdisplays ongmo phological
diffe ences, esembling hechangesal eadyseena 30µM o KOcells.

Figu e5.Cell iabili yandHoechs 33342s aining.Cellswe e ea edwi hdiffe en concen a ions
o H
2
O
2
andexamineda e 8h,24h,48hand72hby esazu inassay.Cells ea edwi hmedium
wi hou supplemen edH
2
O
2
we ede inedas100% iabili y.(A)Cell iabili ya e p io  ea men 
wi h0.3mMBSO;(B)cell iabili ywi hou p io  ea men wi hBSO;(C) luo escenceandb igh 
ilmmic oscopyo HEK-293TMTARC1KOandWTcellsina40×objec i emagni ica ion.Cellswe e
ea ed o 48hwi h30µMH
2
O
2
.Thewhi ea owsma kcellnucleiwi hclea ch oma inconden-
sa ion.
Thisobse a ionwascon i medina esazu in-basedcell iabili yassay.Whilelow
concen a ionso H
2
O
2
dono appea  oha eanega i ein luenceoncell iabili yinei he 
WTo KOcells,wheninc easingH
2
O
2
concen a ionsabo e10µM,WTandKOcells
clea lyshowdi e gence,wi h he iabili yo WTcellsbeingsigni ican lyhighe (Figu e
5A,B).Adec eased iabili yo knockou cellscanal eadybeobse eda e 8handonly
becomese enmo ep onounceda e longe incuba ionpe iods.A e 48h,KOcellsa e
no  iablea 30µMH
2
O
2
,whe eas hesameisobse edwi hWTcellsa aconcen a ion
o 80µMH
2
O
2
.A aconcen a iono 30µM, he iabili yo  heWTcellsiss illapp ox.
70%.
Figu e 5.
Cell iabili y and Hoechs 33342 s aining. Cells we e ea ed wi h di e en concen a ions
o H
2
O
2
and examined a e 8 h, 24 h, 48 h and 72 h by esazu in assay. Cells ea ed wi h medium
wi hou supplemen ed H
2
O
2
we e de ined as 100% iabili y. (
A
) Cell iabili y a e p io ea men
wi h 0.3 mM BSO; (
B
) cell iabili y wi hou p io ea men wi h BSO; (
C
) luo escence and b igh
ilm mic oscopy o HEK-293T MTARC1 KO and WT cells in a 40
×
objec i e magni ica ion. Cells we e
ea ed o 48 h wi h 30
µ
M H
2
O
2
. The whi e a ows ma k cell nuclei wi h clea
ch oma in condensa ion
.
Molecules2023,28,xFORPEERREVIEW6o 12
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
These indingsshow ha H
2
O
2
deg ada ionby hemARC1enzymedoesoccu incell
cul u e,andi hasameasu ableeffec oncellphysiologya highex acellula H
2
O
2
con-
cen a ions.
Fu he ,anin luenceoncellp oli e a ioncouldbeobse ed.Ex acellula concen a-
ionso 10µMH
2
O
2
showednoimpai men oncellp oli e a ion.Anex acellula H
2
O
2

concen a iono 20µMdidlead oimpai edcellp oli e a ion:a e 24h, hep oli e a ion
o bo hWTandKOcellswasdec easedcompa ed ocon olcellswi hou H
2
O
2
 ea men .
Thisimpai men oncellp oli e a ionwasmo ep onouncedinmARC1-de icien cells,
whe e,a e 24h,only50%couldbecoun edcompa ed ocells ea edwi h hemedium
only; hus,inpu elya i hme ical e ms,nocelldi isiono  heKOcellshad akenplacein
helas 24h.Thenumbe o WTcellswas educed oapp ox.70%.A e 72h,bo hWT
andKOcellswe e educed oapp ox.35%.A aH
2
O
2
concen a iono 30µM,nomeasu -
ablecelldi isionoccu edinei he  heKOo WTcells(Figu e6).

Figu e6. Cellp oli e a ion.Cellswe e ea edwi hdiffe en concen a ions(10µM,20µM,30µM)
o H
2
O
2
.A e 24h,48hand72h, hecellnumbe wasde e minedon heImageXp ess
®
(λ
ex
:358,
λ
em
:461)(Molecula De ices,LLC.,SanJose,CA,USA).
3.Discussion
Thes udyp esen edhe eiden i iesH
2
O
2
asanewsubs a e o  hehumanmARC1
andmARC2p o eins.Thedeg ada iono H
2
O
2
wasdemons a edin i owi h ecombi-
nan enzymesandcon i medinamo ecomplexen i onmen byincelluloknockou s ud-
ies.Thus, o  he i s  ime,a educ iono O-Obondsby hemARCenzymesys emis
desc ibed.MolybdenumenzymeslikemARC ypicallycha ac e ize wo-elec on ans e 
eac ions; he e o e, hep oduc o  his eac ionislikelywa e .
Whilewecanno a  hispoin conclude ha H
2
O
2
o o he  eac i eoxygenspeciesa e
hephysiologicalsubs a eo euka yo icmARCenzymes, his indingisimpo an none-
heless.Compoundswi hO-Obondsa eacomple elyno elg oupo po en ialmARC
subs a es ha ha eno p e iouslybeens udied.So a ,allmARC-ca alyzed eac ions
desc ibedin heli e a u ea eN- educ ionsclea ingN-Obonds[18].
The u no e  a eso  hemARC-ca alyzedH
2
O
2
 educ iona e ela i elylowcom-
pa ed oo he H
2
O
2
-deg adingenzymes.I shouldbeno ed,howe e , ha  u no e  a es
de e minedwi h hesoluble ecombinan p o einswi hou  hei OMM-ancho ingse-
quencesa e ypicallymuchlowe  o humanmARCenzymescompa edwi hp o eins
isola ed omo ganhomogena es[19].Thus,in i oH
2
O
2
- educingac i i ieso human
Figu e 6.
Cell p oli e a ion. Cells we e ea ed wi h di e en concen a ions (10
µ
M, 20
µ
M, 30
µ
M)
o H
2
O
2
. A e 24 h, 48 h and 72 h, he cell numbe was de e mined on he ImageXp ess
®
(
λex
: 358,
λem: 461) (Molecula De ices, LLC., San Jose, CA, USA).
Molecules 2023,28, 6384 7 o 12
3. Discussion
The s udy p esen ed he e iden i ies H
2
O
2
as a new subs a e o he human mARC1
and mARC2 p o eins. The deg ada ion o H
2
O
2
was demons a ed
in i o
wi h ecom-
binan enzymes and con i med in a mo e complex en i onmen by in cellulo knockou
s udies. Thus, o he i s ime, a educ ion o O-O bonds by he mARC enzyme sys em is
desc ibed. Molybdenum enzymes like mARC ypically cha ac e ize wo-elec on ans e
eac ions; he e o e, he p oduc o his eac ion is likely wa e .
While we canno a his poin conclude ha H
2
O
2
o o he eac i e oxygen species
a e he physiological subs a e o euka yo ic mARC enzymes, his inding is impo an
none heless. Compounds wi h O-O bonds a e a comple ely no el g oup o po en ial mARC
subs a es ha ha e no p e iously been s udied. So a , all mARC-ca alyzed eac ions
desc ibed in he li e a u e a e N- educ ions clea ing N-O bonds [18].
The u no e a es o he mARC-ca alyzed H
2
O
2
educ ion a e ela i ely low compa ed
o o he H
2
O
2
-deg ading enzymes. I should be no ed, howe e , ha u no e a es
de e mined wi h he soluble ecombinan p o eins wi hou hei OMM-ancho ing sequences
a e ypically much lowe o human mARC enzymes compa ed wi h p o eins isola ed om
o gan homogena es [
19
]. Thus,
in i o
H
2
O
2
- educing ac i i ies o human mARC enzymes
can be expec ed o be signi ican ly highe han he alues epo ed he e. On ano he no e,
he K
M
alues o H
2
O
2
educ ion by ecombinan human mARC p o eins (app ox. 50
µ
M)
a e e y low compa ed o hose o he well-s udied N-hyd oxyla ed compounds.
Hyd ogen pe oxide has undamen ally impo an unc ions in humans. Depending
on he in acellula concen a ion, i ini ia es, in e alia, cell p oli e a ion, cell shaping,
mig a ion and angiogenesis [
20
–
22
]. On he o he hand, he accumula ion o highe con-
cen a ions o hyd ogen pe oxide and o he ROS leads o oxida i e s ess, a condi ion o
imbalance be ween p o-oxidan s and an ioxidan s. ROS pass h ough cell memb anes and
cause oxida i e damage o lipids, p o eins and DNA, as well as mi ochond ial dys unc ion,
all o which can lead o he loss o essen ial cell unc ions and ini ia e he caspase-media ed
apop osis pa hway [23–25].
In his s udy, a coupled enzyme assay was es ablished. Two pa ame e s we e mea-
su ed: he amoun o NADH oxidized by he mARC-media ed educ ion and, o con i m
he esul s, he emaining concen a ion o hyd ogen pe oxide. Thus, i was shown ha
bo h mARC p o eins can educe H2O2.
Conside ing he enzyme kine ics o his eac ion, i is s iking ha he K
M
alues o
bo h mARC p o eins a e ema kably lowe when compa ed o well-known H
2
O
2
-deple ing
enzymes such as ca alase o pe oxi edoxin [26,27].
An in cellulo MTARC1 knockou model was gene a ed and es ablished o e i y
whe he he absence o mARC1 leads o cellula impai men upon exposu e o H
2
O
2
. A
signi ican ly educed cell physiology o mARC1-de icien cells and hus a highe sensi i i y
owa d H2O2compa ed o co esponding WT cells could be obse ed. Also, highe apop-
osis le els and lowe cell iabili y le els we e seen. These indings could be con i med by
ligh and luo escence mic oscopy showing al e ed cell mo phology and declined nuclea
condensa ion. While he H
2
O
2
concen a ions used in ou cell cul u e expe imen s ce ainly
exceed hose expec ed
in i o
, i is s ill possible ha he egula ion o ROS is a physiological
unc ion o mARC enzymes.
The complex mechanisms o hyd ogen pe oxide egula ion wi h a la ge numbe o
enzymes in a ious cell o ganelles demons a e he need o di e en app oaches o con ol
he in acellula concen a ion. While majo hyd ogen pe oxide ans o ming enzymes like
ca alase, GPx o pe oxi edoxins a e p esen in he endoplasmic e iculum, cy osol, nucleus,
pe oxisomes, he in e memb ane space (IMS), inne mi ochond ial memb ane (IMM) and
mi ochond ial ma ix, mARC s ands ou h ough i s localiza ion a he OMM [
13
,
15
,
28
–
30
],
al hough he e a e some epo s abou GPx also being localized a he OMM [31].
mARC migh hus be in ol ed in p o ec ing he OMM om ROS. Since ROS a e
o med in high concen a ions in he IMS and he cy osol by di e en enzymes, a p o ec-
i e mechanism o he undesi ed oxygena ion o he OMM— o example, agains lipid
Molecules 2023,28, 6384 8 o 12
pe oxida ion o p e en oxida i e s ess and mi ochond ial dys unc ion—is concei able.
This was also desc ibed ea lie o GPx-4 a he IMM [
28
]. The e a e also some known
enzymes a he OMM, such as he monoamine oxidase MAO, ha o m hyd ogen pe oxide
as a seconda y p oduc [
28
]. I is hus also possible ha mARC in luences he ee di usion
be ween IMS and cy osol di usion and he anspo o H
2
O
2
h ough ol age-dependen
anion channels (VDACs) and pe oxipo ins due o i s high a ini y o H2O2.
Kagan and colleagues highligh ed he signi icance o ROS o apop osis by iden i-
ying he eleasing pa hway o p oapop o ic ac o s om he OMM. A H
2
O
2-
dependen
ca diolipin-speci ic pe oxidase ac i i y o cy och ome c is equi ed o he pe meabiliza ion
o he OMM, demons a ing again he signi icance o hyd ogen pe oxide egula ion in he
OMM o c i ical cell p ocesses [
32
]. Also, H
2
O
2
is o med in he pe oxisomal
β
-oxida ion
o a y acids [
33
]. Va ious s udies in mice and a s also sugges he possibili y o a dual
localiza ion o mARC in mi ochond ia and pe oxisomes [
34
,
35
]; hus, his colocaliza ion
could sugges ha mARC has a egula o y unc ion in hyd ogen pe oxide and an ioxidan
me abolism in pe oxisomes as well.
In conclusion, he educ ion o H
2
O
2
by mARC is ce ainly e y in e es ing, as i
indica es ha he spec um o subs a es ha hese enzymes can educe could go a
beyond he p e iously s udied N-oxygena ed subs a es. The in cellulo s udies con i m
ha H
2
O
2
is also educed by he na i e mARC enzyme in i s cellula con ex . We do no
claim ha H
2
O
2
o o he eac i e oxygen species a e he physiological subs a es o mARC.
An in ol emen in he cellula egula ion o H
2
O
2
is concei able, bu da a a ailable on his
poin a e no su icien o claim his o be he enzymes’ unc ion. Due o hei in ol emen
in li e disease, mARC enzymes ha e ecen ly gained much a en ion. Howe e , i emains
unknown wha he physiological unc ion o mARC ac ually is and how exac ly i exe s
i s in luence on lipid me abolism and li e disease. In he u u e, a sea ch o mARC’s
physiological subs a e ha migh no ha e p e iously been associa ed wi h mARC should
be conside ed.
4. Ma e ials and Me hods
4.1. P o ein Sou ces
Recombinan human mARC1, mARC2, Cyb5B and Cyb5R3 we e exp essed in
Esche ichia coli (E. coli) and pu i ied by column ch oma og aphy, essen ially as desc ibed p e-
iously [
36
]. Fo mARC1 and mARC2 wi h bound molybdop e in co ac o (holo-mARC1/2),
he E. coli TP1000 s ain [
37
] was used. P o eins wi hou molybdop e in we e exp essed
in RK5202 [
38
]. P o ein concen a ions we e de e mined using he Pie ce BCA P o ein
Assay Ki (The mo Fishe Scien i ic, Wal ham, MA, USA) wi h bo ine se um albumin o
calib a ion. Loading o Cyb5B wi h heme and Cyb5R3 wi h la in adenine dinucleo ide
(FAD) was quan i ied as published [36].
4.2. Pho ome ic Assay
Reduc ion o H
2
O
2
by he econs i u ed mARC enzyme sys em was assayed using he
p e iously published p o ocol [
2
]. Reac ions con ained 7.5
µ
g (224 pmol) o ei he mARC1
o mARC2, 3.5
µ
g (210 pmol) Cyb5B and 0.08
µ
g (2.4 pmol) Cyb5R3 and 200
µ
M NADH
in 20 mM Na-MES bu e , pH 6.0. The o al eac ion olume was 300
µ
L. Consump ion
o NADH a 37
◦
C was moni o ed by eco ding he abso p ion spec um om 300 o
400 nm in 15 s in e als. The eac ion was s opped by hea ing 200
µ
L o he incuba ion mix
o 95
◦
C o 5 min in a wa e ba h. Tu no e a es we e calcula ed h ough he change in
abso p ion a 340 nm o e a imespan o 2 min. Kine ic pa ame e s we e de e mined by
i ing he Michaelis–Men en equa ion o he u no e a es a di e en H
2
O
2
concen a ions
in G aphPad P ism 9.5.1. All measu emen s we e pe o med in iplica e.
4.3. Fluo ome ic Ac i i y Assay
Al e na i ely, he enzyme ac i i y was assayed by moni o ing NADH consump ion
h ough a ecen ly es ablished luo ome ic p o ocol [
17
]. B ie ly, ime-dependen change
Molecules 2023,28, 6384 9 o 12
in NADH luo escence (
λex
= 340 nm;
λem
= 365 nm) was moni o ed wi h a TECAN In ini e
200 M P o pla e eade . The eac ion olume was 50
µ
L. Assays con ained 193 pmol
(=6.5
µ
g) hmARC-1, 65 pmol hCyb5B (heme), 6.5 pmol hCyb5R3 (FAD), 0.2 mM o NADH
and he subs a e o be es ed in 20 mM Na-MES bu e , pH 6.0. The eac ion mix u es
con aining all componen s excep Cyb5R3 we e p e-incuba ed a 37
◦
C o 3 min. The
eac ions we e s a ed by adding Cyb5R3, and NADH luo escence was eco ded o
15 min a 37 ◦C. BAO was always used in pa allel as a e e ence subs a e.
4.4. Pe oxide Assay
To con i m deg ada ion o H
2
O
2
by he mARC enzyme sys em, esidual H
2
O
2
con-
cen a ions we e quan i ied using a luo ome ic pe oxidase assay [
39
]. Samples om he
pho ome ic ac i i y assays we e cooled on ice o 1 min and hen p e-incuba ed a 37
◦
C o
2 min. Then, 10
µ
L o 20 mM Na-MES bu e , pH 6.0, 30
µ
L o 20 mM 4-hyd oxyphenylace ic
acid and 30
µ
L o a 30
µ
g/mL ho se adish pe oxidase solu ion we e added and incuba ion
a 37
◦
C was con inued o 10 min. A e wa ds, 10
µ
L o 10 M NaOH and 850
µ
L dis illed
wa e we e added. A o al o 150
µ
L was ans e ed o Pe kin Elme qua z SUPRASIL
cu e es. Fluo escence spec a om 340 o 450 nm we e measu ed in a Pe kin Elme LS
55 Fluo escence Spec ome e using an exci a ion wa eleng h o 320 nm. The peak a
408 nm was used o e alua ion. Co ela ion be ween he in ensi y o his peak and he
H2O2concen a ion was p o en using a calib a ion cu e.
4.5. Molecula Biology
Knockou o he MTARC1 gene in HEK-293T cells was achie ed by he CRISPR-
Cas9 me hod [
40
]. A sequence encoding sgRNA o sgRNA add essing exon 2 o he
MTARC1 gene (5
0
-GTGGCCAAAACCGAACACTAGT-
TGG
-3
0
, PAM sequence unde lined)
was cloned in o he Esp3I si e o he plen iCRISPR 2 plasmid (Addgene #49535) using
s anda d cloning me hods [
41
]. Co ec inse ion o he sgRNA-encoding sequence was
con i med by Sange sequencing using he p ime 5
0
-GAGGGCCTA-TTTCCCATGATTCC-3
0
.
4.6. Mammalian Cell Cul u e
Human emb yonic kidney cells (HEK-293T) we e g own in Dulbecco’s Modi ied
Eagle Medium (DMEM) supplemen ed wi h 10% e al cal se um (FCS) in a humidi ied
incuba o a 37
◦
C in p esence o 5% CO
2
. HEK-293T cells we e e i ied by SNP analysis
and con i med o be mycoplasma- ee.
Fo ans ec ion, a ge cells we e seeded a 2
×
10
5
cells/well in a 6-well pla e. Twen y-
ou hou s a e seeding, he cul i a ion medium was eplaced wi h DMEM con aining
2% FCS. The ans ec ion mix consis ed o 100
µ
L Op i-MEM, 1
µ
g DNA (plen iCRISPR 2
con aining he sgRNA sequence) and 3
µ
L Lipo ec amine 2000
®
(The mo Fishe Scien i ic,
Wal ham, MA, USA). Medium was exchanged o DMEM incl. 10% FCS a e 6 h. A e u -
he 18 h, medium was eplaced again by DMEM incl. 10% FCS, supplemen ed wi h
2.5
µ
g/mL pu omycin. Cells we e cul i a ed and selec ed in pu omycin-con aining
medium o 6 days.
HEK-293T KO lines we e isola ed by se ial dilu ion in 96-well pla es (0.5 cells/well).
A e h ee weeks o expansion, DNA was isola ed wi h he peqGOLD mic ospin issue
DNA ki (VWR, Da ms ad , Ge many), he egion o in e es was ampli ied by PCR and he
knockou was alida ed by Sange sequencing. P ime s o bo h ampli ica ion o he gene
egion o in e es as well as sequencing we e 5
0
-AAGCTCCTCCAGGGTCTGGCTTC-3
0
and e e se 50-CGACCTGCCCTTTCCTTACCTGC-30.
Fo immunoblo analysis, cells we e de ached using ice-cold Dulbecco’s PBS (DPBS),
cen i uged, and esuspended in NP-40 lysis bu e (con aining 150 mM NaCl, 1% ( / )
Nonide P-40, 50 mM T is). A e 30 min shaking a 4
◦
C, he lysa e was cen i uged again
and he p o ein concen a ion in he supe na an was quan i ied using he Pie ce BCA
P o ein Assay Ki (The mo Fishe Scien i ic).