25th International Meeting on Organic Geochemistry IMOG 2011

O-27
Coenzyme factor 430: abundance and isotopic compositions for
tracing a key molecule of methanogenesis and reversemethanogenesis
Yoshinori Takano, Hiroyuki Imachi, Nana O. Ogawa, Yoshito Chikaraishi, Nao Ohkouchi
Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan
(corresponding author:takano@jamstec.go.jp)
Introduction
Coenzyme Factor 430 (F430) is the prosthetic group
of methyl-coenzyme M reductase (MCR) and the key
enzyme in biological methane-forming step in
methanogenic archaea and methane-oxidizing step in
methanotrophic archaea [1-3]. The structure of F430
has been established based on spectroscopic studies
of isolated coenzyme and its partial synthetic
derivatives. The coenzyme is a yellow non-fluorescent
nickel hydroporphyrin having unique pentacarboxyl
structure (Figure 1: the reduced Ni(I) is active state for
the enzyme). We have recently established a method
to identify and quantify F430 in microbial slurry with its
compound-specific carbon and nitrogen isotopic
compositions for the applications of methane
biogeochemistry and global carbon cycle.
Experimental
We extracted yellowish F430 fraction (Fig.1) from
archaeal culture samples (methanogen assemblages,
e.g., Methanobacterium, Methanosarcina, and ANME
assemblages in sludge environments). Then, we
isolated F430 from the fraction by two steps column
chromatography (QAE Sephadex A25 and XAD (PAD
I) resin: [4]) and further purified by reversed-phase
high performance liquid chromatography/electrospray
ionization mass spectrometry (HPLC/ESI-MS: Agilent
1100) on the gradient program. The base-line
resolution of F430 was validated by photo diode array
detector (DAD), total ion chromatogram (TIC) and
selected ion monitoring (SIM). The carbon and
nitrogen isotopic compositions of the purified F430
and bulk cell were determined by an isotope ratio
mass spectrometer (IRMS; ThermoFinnigan Delta
plus XP) coupled with a Flash elemental analyzer
(EA; ThermoFinnigan EA1112) via a Conflo III
interface. We also conducted 16S rRNA gene-based
clone analysis for the archaeal culture samples.
Results and Discussion
Figure 1 shows 3-D chromatogram of the isolated
F430, whose purity is high enough for compoundspecific
isotope analysis. F430 is about 10‰ depleted
in 13 C relative to bulk microbial slurry on average. This
13 C depletion in F430 suggests that the methanogen
13
are substantially C-depleted relative to other
microbes. We estimated F430 abundance of 600-900
nmol/dry-cell gram in the methanogen. Although we
are still in a preliminary stage, we suggest that the
present method is useful for estimating activities of
methanogenesis and reverse-methanogenesis in the
sub-seafloor environment. Furthermore, carbon and
nitrogen isotopes of F430 will provide information on
the processes mediated by methanogens and
anaerobic methanotrophs.
References
[1]Thauer, R. (1998) Biochemistry of
methanogenesis. Microbiology, 144, 2377-
2406.
[2]Kruger, M. et al. (2003) A conspicuous nickel
protein in microbial mats that oxidize methane
anaerobically. Nature, 426, 878-881.
[3]Scheller, S. et al. (2010) The key nickel enzyme of
methanogenesis catalyses the anaerobic
oxidation of methane. Nature, 465, 606-608.
[4]Mayr, S. et al. (2008) Structure of an F430 variant
from archaea associated with anaerobic
oxidation of methane. J. Amer Chem Soc, 130,
10758-10767.
Figure 1 Online RP-HPLC/ESI-MS separation of the
nickel porphinoid, coenzyme F430 (C42H51N6NiO13),
from methanogen.
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