Metal Reduction by Methanobactins
Teresa Swanson1, Lisa Chesner1, Leann Seidl1, Elizabeth Casper1, Alan A. Dispirito3 PhD, Jeremy Semrau2 PhD, Warren Gallagher1 PhD, Scott Hartsel1 PhD
1University
of Wisconsin-Eau Claire, Eau Claire WI USA 2University of Michigan, Ann Arbor MI USA 3Iowa Sate University, Ames IA USA
ABSTRACT
A.
Methionine: susceptible to oxidation. However, this amino
acid is not present in all samples of MB and not at all in SB2.
B.
Tyrosine: can form a stable free radical in some biological
situations. This would broaden the NMR spectra, which is not
observed. Tryosine is also not seen in SB2.
A.
INTRODUCTION TO METHANOBACTINS
Methanotrophs are part of the global carbon cycle and are major
players in bioremediation. Their methane reducing enzymes
require large amounts of copper. Methanotrophs secrete a small
Cu binding peptide, methanobactin1,4, which has highly unusual
functional groups, including two oxazalone rings and thioamides in
the Cu binding site. Recent evidence suggests that methanobactinlike molecules may be more common than previously thought,
even possibly occurring in eukaryotes3. An unexplained feature of
MB is the mechanism by which is reduces Cu(II) and stabilizes Cu(I)
in an aqueous environment. The recent purification of a new
species of methanobactin, SB2, allows us to test hypotheses
concerning Cu-reduction and stabilization. SB2 is quite distinct
from MB, lacking cysteines, methionines, and tyrosines while
retaining the thioamides and oxazalone ring.
Cysteine residues: could provide 2 electrons in a change from
the free thiol form (-SH, not as shown) to a –S-S- bond with
no free radical. There is no evidence for free sulfhydryls in
MB, as shown by mass spectra (which are identical between
Cu(I) and a lack of reaction of MB with N-Ethylmaleimide.
NEM reacts with free sulfhydryl groups. If these
sulfhydryls were important for the reduction of
copper, there would be a difference in the
absorbance spectra. A new MB-NEM adduct is
not observed. The difference in the spectra is
only due to the absorbance of the NEM and not a
reaction with methanobactin.
D
B.
Water3: Forcing Cu(II) in tetrahedral binding environment
may give it a higher reduction potential than H2O/O2 at ~
+0.8V. Problem: The free radical left over from H2O would be
a hydroxyl radical, OH•. Two hydroxyl radicals could be
released as H2O2 from a MB tetramer, but no evidence of
this is seen.
C.
Thioamides2: Thiourea is known to reduce Cu(II) to Cu(I). MB
contains 2 similar thioamides.
302nm
1:2 Cu:Mb is the ratio of the saturation
point seen in the absorbance spectra of
MB with Cu(II).
B
A
0.9
0.7
0.6
Absorbance
0 Cu
0.1 Cu
0.2 Cu
0.3 Cu
0.4 Cu
0.5 Cu
0.6 Cu
0.7 Cu
0.8 Cu
0.9 Cu
1.0 Cu
1.25 Cu
1.5 Cu
1.75 Cu
2.0 Cu
UV/VIS spectrum of MB
titrated with CuSO4(aq) to
saturation
0.8
0.5
0.4
0.3
0.2
0.1
0
250
300
350
400
Wavelength (nm)
(I)
2Cu2+ + 3MB  2 MBCu+ + Product A
2:3 Cu:MB
(II)
2Cu2+ + 4 MB  2 MBCu+ + Product B
1:2 Cu:MB
Product A
Product B
Chemical Formula: C90H112N20O32S102Exact Mass: 2304.4970
Molecular Weight: 2306.6182
M/Z -1: -2306.6182
M/Z -2: -1153.3091
Chemical Formula: C45H55N10O16S5Exact Mass: 1151.2407
Molecular Weight: 1152.3011
M/Z -1: -1152.3011
M/Z -2: -576.15055
282nm
340nm
E2
C
We have eliminated the amino acid residues as internal
reductants through various techniques and comparison to SB2.
Also, the improbability of water as an electron source leads us to
a different theory based on well-known thiourea observations.
There are two possibilities for this “Sacrificial Twin Theory”.
Product B shows a 1:2 Cu:Mb ratio which is also the ratio of the
saturation point seen in the absorbance spectra of MB with Cu(II).
The cleanliness of the NMR spectra suggests that all the MB share the
same environment, however, >1:2 Cu:MB is the ratio at which
broadening of the NMR spectra occurs due to incomplete reduction.
These data support the Sacrificial Twin Theory in which two MB
would be cross-linked to form a dimer: two “sacrificed” MB so that
the other two may use the electrons to reduce copper.
254nm
E1
DISCUSSION
ELIMINATING INTERNAL REDUCTANTS
Methanobactins (mb) are copper-binding peptides or chalkophores secreted by
methanotrophs to scavenge copper ions from the environment. A unique feature of these
molecules is their ability to reduce and stabilize copper as Cu(I). In the absence of Cu, the
molecule will also bind and reduce many other metals including silver, gold and mercury.
We have investigated the methanobactin (MB) from Methylosinus trichosporium OB3b,
as well as a newly isolated methanobactin from Methylocystis sp. SB2 in order to
determine how these novel polypeptides reduce and stabilize Cu(I) and other metals. We
have used UV-Visible, NMR, and LC-TOF to characterize both methanobactins in an
attempt to find the internal reductant, determine the process of reduction, and look for
structural similarities. Our results eliminate methionine, cysteine and tyrosine as reducing
agents for Cu(II) and TOF mass spectra show no difference between MB exposed to Cu(I)
vs. Cu(II). The most likely remaining sources of electrons are the thioamides or water
which would have to be oxidized by a Cu(II) with an unusually high reduction potential.
450
500
550
ACKNOWLEDGEMENTS
NSF-MRI Grant CHE 0521019 to UW-Eau Claire for the purchase of a Bruker Avance II 400MHz NMR
Spectrometer. NSF-MRI Grant CHE 0619296 to UW-Eau Claire for the purchase of an Agilent 6210 ESITOF LC/MS UW- Eau Claire Office of Research and Sponsored Programs
We have not yet seen much evidence to support the Sacrificial Twin
Theory but future research will look for products with the masses of
A or B in TOF-MS and will also involve the search for other species of
methanobactins, as well as their overall role in the global copper
cycle.
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