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. REFERENCES 1 Behling, Lee, Hartsel, Scott, Lewis, David, DiSpirito, Alan, Choi Dong, Masterson, Larry, Veglia, Gianluigi, Gallagher, Warren, “NMR, Mass Spectrometry and Chemical Evidence Reveal a Different Chemical Structure for Methanobactin That Contains Oxazolone Rings” Journal of the American Chemical Society (2008) 130:38 1260412605. 2Belle, Catherine, Rammal, Wassim, Pierre, Jean-Louis. “Sulfur Ligation in Copper Enzymes and Models.” Journal of Inorganic Biochemistry (2005) 99: 1929-1936. 3Cobine, A. Paul, Pierrel, Fabien, Bestwick, L. Megan, Winge, R. 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