Si-face stereospecificity at C5 of coenzyme F
420
for F
420
H
2
oxidase from methanogenic Archaea as determined by
mass spectrometry
Henning Seedorf
1
,Jo
¨
rg Kahnt
1
, Antonio J. Pierik
2
and Rudolf K. Thauer
1
1 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
2 Fachbereich Biologie, Philipps-Universita
¨
t, Marburg, Germany
Methanogenic Archaea fluoresce greenish yellow when
irradiated with UVA light. The fluorescence is due to
coenzyme F
420
, a 7,8-didemethyl-8-hydroxy-5-deaza-
riboflavin derivative (Fig. 1). The coenzyme, which is
generally present in 1 mm intracellular concentrations
[1], functions as a redox mediator in methanogenesis,
in NADP

-dependent enzymes analysed to date in this
respect have been shown to be Si-face stereospecific at
C5 of F
420
[6]. This is surprising because NAD(P)-
dependent enzymes can be Si-face or Re-face specific
[7] and some flavoenzymes, whose apoproteins catalyse
the reduction of synthetic 8-hydroxy-5-deaza-FAD,
have been shown to be Si-face stereospecific with
respect to C5 of the synthetic deazaflavin and others
to be Re-face stereospecific [7,8]. In case of the
pyridine-nucleotide-dependent enzymes the redox
potential (E°) of the electron acceptor reduced by
NAD(P)H is thought to be an important factor deter-
mining the stereospecificity of these enzymes [9,10].
Keywords
coenzyme F
420
; 5-deazaflavins;
F
420
H
2
oxidase; methanogenic Archaea;
stereospecificity
Correspondence
R. K. Thauer, Max Planck-Institute for
Terrestrial Microbiology, Karl-von-Frisch-
Strasse, D-35043 Marburg, Germany
Fax: +49 642 117 8109

H
2
with O
2
to [5-
1
H]F
420
rather than to
[5-
2
H]F
420
as determined by MALDI-TOF MS. (5S)-[5-
2
H
1
]F
420
H
2
was
generated by stereospecific enzymatic reduction of F
420
with (14a-
2
H
2
)-
[14a-

-dependent formate dehydrogenase; Frh,
F
420
-reducing hydrogenase; H
4
MPT, tetrahydromethanopterin; Mer, F
420
-dependent methylenetetrahydromethanopterin reductase;
methylene-H
4
MPT, methylenetetrahydromethanopterin; Mtd, F
420
-dependent methylenetetrahydromethanopterin dehydrogenase; TFA,
trifluoroacetic acid.
FEBS Journal 272 (2005) 5337–5342 ª 2005 FEBS 5337
Redox potentials (E°) of the electron acceptor more neg-
ative than )200 mV generally promote Si-face stereo-
specificity and redox potentials more positive than
)200 mV promote Re-face stereospecificity at C4
of NAD(P). Thus the NADP-dependent glucose-6-phos-
phate dehydrogenase is Si-face specific (E° ¼
)330 mV) and the NAD-dependent malate dehydro-
genase is Re-face specific (E° ¼ )170 mV). Most eth-
anol dehydrogenases are Re-face specific (E° ¼
200 mV), but the enzyme from Drosophila melanogaster
is Si-face specific. For recent literature on the subject see
Berk et al. [11].
The following eight F
420
-dependent enzymes have

-dependent glucose-6-phosphate de-
hydrogenase (Fgd) [6]. Adf, Mer and Fgd form a fam-
ily, as do the F
420
-binding subunits FpoF, FrhB and
FrdB. The two families, Mtd and Fno are not phylo-
genetically related. The eight enzymes catalyse redox
reactions with electron acceptors ranging in redox
potential (E°
¢
) from )414 mV (2H
+
⁄ H
2
)to)165 mV
(methanophenazine ox ⁄ red) [19]. The Si-face stereospe-
cificity of F
420
-dependent enzymes thus appears to be
independent of their phylogenetic origin and of the
thermodynamics of the reactions catalysed by them.
Recently a novel F
420
-dependent enzyme, F
420
H
2
oxidase (FprA), was discovered in methanogenic Arch-
aea [20]. FprA catalyses the oxidation of 2 F
420

investigated the stereospecificity of this enzyme and
found it to be Si-face specific at C5 of F
420
. The
method used was based, in principle, on the technique
for determining the hydride transfer stereospecificity of
nicotinamide adenine dinucleotide-linked oxidoreduc-
tases by MS [21].
Results
The following findings are important for the under-
standing of the results shown in Fig. 2: (a) [5-
1
H]F
420
and [5-
2
H]F
420
can be identified and the relative
amounts present in a mixture quantitated using
MALDI-TOF-MS; (b) F
420
H
2
auto-oxidizes nonstereo-
specifically to F
420
in the matrix used for MALDI-
TOF-MS; (c) F
420

1
H]F
420
(Fig. 2Aa); the spectrum of [5-
1
H
2
]F
420
H
2
generated from [5-
1
H]F
420
by Mtd-catalysed reduction
with [14a-
1
H
2
]methylene-H
4
MPT (Fig. 2Ab); and the
spectrum of [5-
1
H]F
420
generated from [5-
1
H

17
O and
18
O.
The experiment shown in Fig. 2B differs from that
in Fig. 2A only in that in the first step F
420
was enzy-
matically reduced with [14a-
2
H
2
] methylene-H
4
MPT
yielding (5S)-[5-
2
H
1
]F
420
H
2
. FprA catalysed oxidation
of (5S)-[5-
2
H
1
]F
420

and [5-
2
H]F
420
, as indicated by the relative
intensities of the 772 and 773 Da mass peaks
Fig. 1. Structure of reduced coenzyme F
420
(F
420
H
2
). F
420
¼ N-(N-
L-lactyl-L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-
8-hydroxy-5-deazariboflavin.
Stereochemistry of F
420
H
2
oxidase H. Seedorf et al.
5338 FEBS Journal 272 (2005) 5337–5342 ª 2005 FEBS
(Fig. 2Bb, stick spectrum, black). For comparison the
relative intensities calculated for a 1 : 1 mixture are
given (Fig. 2Bb, stick spectrum, white). The [5-
1
H]F
420
to [5-

2
. The FprA-catalysed oxidation of
the mixture yielded a 1 : 1 mixture of [5-
1
H]F
420
and
[5-
2
H]F
420
as revealed by the relative intensities of the
772 and 773 Da mass peaks (Fig. 2Cc). The results are
consistent with FprA catalyzing the oxidation of (5S)-
[5-
2
H
1
]F
420
H
2
to [5-
1
H]F
420
and the oxidation of (5R)-
[5-
2
H

420
, despite four of these
enzymes being unrelated phylogenetically. The finding
that F
420
H
2
oxidase (FprA) is also Si-face specific brings
to five the number of Si-face-specific F
420
-dependent
enzymes that are not related phylogenetically. There is
only a 6.25% probability that this is by chance.
To date, the crystal structures of four F
420
-dependent
enzymes have been resolved: F
420
H
2
:NADP oxido-
reductase, with and without F
420
bound [22];
F
420
-dependent alcohol dehydrogenase with F
420
bound
[23]; Mer, with and without F

hydrogenase. (A) Experiment with nonlabelled substrates showing
that the mass spectrum of [5-
1
H
2
]F
420
H
2
(b), owing to auto-oxida-
tion of F
420
H
2
, is identical to that of [5-
1
H]F
420
(a, c). The simulated
stick spectra (white) are for [5-
1
H]F
420
. (B) Experiment with specif-
ically
2
H-labelled substrates showing that the mass spectrum of
F
420
formed from (5S)-[5-

. The other two (a, c) are for [5-
1
H]F
420
.(C)
Experiment with NaB
2
H
4
-reduced [5-
1
H]F
420
showing that the mass
spectrum of F
420
formed from reduced F
420
by FprA-catalysed oxi-
dation corresponds to that of a mixture of [5-
1
H]F
420
and [5-
2
H]F
420
(c). The simulated stick spectrum of reduced F
420
(b, white) and

conformation of methylenetetrahydromethanopterin
and methylenetetrahydrofolate for the dehydrogenation
from the Re-face is energetically favoured [28]. How-
ever, in the case of F
420
, there are no centres of asym-
metry in the near neighbourhood of C5 that could
interact with the reactant or the product, or affect the
transition state(s) and by that induce an intrinsic ener-
getic difference in the reaction profiles involving the Si
versus the Re side of F
420
. The nearest asymmetry cen-
tres are in the N10 side chain. It is therefore difficult
to envisage how the Si-face stereospecificity of F
420
-
dependent enzymes could be dictated by the structure
of F
420
.
Experimental procedures
Isotopes, coenzymes and enzymes
Deuterium oxide (
2
H
2
O) and deuterated formaldehyde
(
2

2
H
2
]methylene-H
4
MPT by spontaneous
reaction of H
4
MPT and
2
H
2
CO [30]. FprA from M. marbur-
gensis [20] and Mtd from Methanopyrus kandleri [26] were
produced heterologously in Escherichia coli and purified to
specific activities of 100 and 4000 UÆmg
)1
, respectively
(1 U ¼ 1 lmolÆmin
)1
). Protein was determined with the Rot-
Nanoquant-Microassay from Roth (Karlsruhe, Germany)
using bovine serum albumin as standard.
Assay to determine the stereospecificity of F
420
H
2
oxidase
The assay is described in Fig. 2B. The 1.2 mL assay mix-
ture at 30 °C contained 60 lm H

H
2
CO) was started by the addition of 120 U Mtd (Si-face
specific) and was completed after 5 min. Subsequently,
60 U FprA were added, which catalysed the oxidation of
F
420
H
2
with O
2
as the electron acceptor. Samples of the
assay were taken before and 5 min after the addition of
Mtd and 5 min after the addition of FprA and analysed by
MALDI-TOF-MS.
In the control experiment described in Fig. 2A, the
1.2 mL assay mixture contained 140 lm
1
H
2
CO instead of
140 lm
2
H
2
CO.
In the control experiment described in Fig. 2C, the
1.2 mL assay did not contain H
4
MPT, H

420
H
2
by MALDI-TOF-MS
Samples (25 lL) of the 1.2 mL assay mixtures were applied
to a small ZipTips (Millipore Corp, Bedford, MA, USA)
column previously equilibrated with 0.1% (v ⁄ v) trifluoro-
acetic acid (TFA). The column was then washed with 0.1%
(v ⁄ v) TFA to remove salts and was then eluted with 84%
(v ⁄ v) acetonitrile ⁄ 0.1% (v ⁄ v) TFA. The eluate was dried by
vacuum centrifugation and the dried pellet dissolved in
10 lL 0.1% (v ⁄ v) TFA and subsequently supplemented
with 10 lL of a saturated solution of a -cyano-4-hydroxy-
cinnamic acid in 70% (v ⁄ v) acetonitrile ⁄ 0.1% (v ⁄ v) TFA.
Aliquots were air dried and analysed by MALDI-TOF-MS.
The mass spectra were collected in the reflector negative-
ion mode. For each spectrum, at least 150 single shots were
summed. The spectra were determined with a Voyager DE
RP from PE Biosystems.
The natural isotopic distribution in F
420
was calculated
by the isotope pattern calculator provided by the University
of Sheffield at the ChemPuter site (http://www.shef.ac.uk/
chemistry/chemputer/). All calculations of simulated data
were carried out in excel 2000 and transformed into stick
spectra separated by 1 Da [31].
Acknowledgements
This work was supported by the Max Planck Society
and by the Fonds der Chemischen Industrie. Henning

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