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Protein Complexing in a Methanogen Suggests Electron Bifurcation and Electron Delivery from Formate to Heterodisulfide Reductase

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Protein Complexing in a Methanogen Suggests Electron Bifurcation and Electron Delivery from Formate to Heterodisulfide Reductase Protein complexing in a methanogen suggests electron bifurcation and electron delivery from formate to heterodisulfide reductase Kyle C. Costaa,b, Phoebe M. Wonga, Tiansong Wanga,c, Thomas J. Liea, Jeremy A. Dodswortha,b,1, Ingrid Swansona, June A. Burna, Murray Hackettc, and John A. Leigha,b,2 aDepartment of Microbiology, bNational Science Foundation Integrative Graduate Education Research Traineeship Program in Astrobiology, and cDepartment of Chemical Engineering, University of Washington, Seattle, WA 98195 Edited by William Metcalf, University of Illinois, Urbana, IL, and accepted by the Editorial Board May 6, 2010 (received for review March 19, 2010) In methanogenic Archaea, the final step of methanogenesis gen- Most methanogens can use H2 as the electron donor, and many fi erates methane and a heterodisul de of coenzyme M and co- can also use formate. Reduced coenzyme F420 (F420H2)isarequired fi enzyme B (CoM-S-S-CoB). Reduction of this heterodisul de by intermediate, and can be generated from H2 by F420-reducing hy- fi heterodisul de reductase to regenerate HS-CoM and HS-CoB is an drogenase (Fru) or by a cycle involving the enzymes H2-dependent Nat Rev Micro- exergonic process. Thauer et al. [Thauer, et al. 2008 methylene-H4MPT dehydrogenase and F420-dependent methylene- biol – 6:579 591] recently suggested that in hydrogenotrophic metha- H4MPT dehydrogenase (2). When formate is the electron donor, fi nogens the energy of heterodisul de reduction powers the most it is oxidized to CO2 by a formate dehydrogenase (Fdh) that endergonic reaction in the pathway, catalyzed by the formylmetha- yields F420H2. nofuran dehydrogenase, via flavin-based electron bifurcation. Here How net energy is conserved in most methanogens is not well we present evidence that these two steps in methanogenesis understood, because the membrane potential generated during are physically linked. We identify a protein complex from the the methyl transfer from H4MPT to HS-CoM would appear to be Methanococcus maripaludis hydrogenotrophic methanogen, , that depleted by Eha to fuel the reduction of CO2 to formyl-MFR. contains heterodisulfide reductase, formylmethanofuran dehydro- The solution to this dilemma apparently resides in the exergonic genase, F420-nonreducing hydrogenase, and formate dehydroge- heterodisulfide reduction step. Methanogens from the order nase. In addition to establishing a physical basis for the electron- Methanosarcinales, known as the methylotrophic methanogens, bifurcation model of energy conservation, the composition of the have a membrane-bound electron transport chain involving the complex also suggests that either H2 or formate (two alternative quinone-like methanophenazine and a cytochrome-containing Hdr electron donors for methanogenesis) can donate electrons to the complex that translocates protons across the cell membrane con- fi heterodisul de-H2 via F420-nonreducing hydrogenase or formate comitant with CoM-S-S-CoB reduction, resulting in net energy fl via formate dehydrogenase. Electron ow from formate to the het- conservation (1). However, all other methanogens (the hydro- fi erodisul de rather than the use of H2 as an intermediate represents genotrophic methanogens) lack methanophenazine and cyto- fl a previously unknown path of electron ow in methanogenesis. We chromes, have a cytoplasmic Hdr, and are not known to generate further tested whether this path occurs by constructing a mutant a membrane potential at this step (1). Nevertheless, these organ- lacking F420-nonreducing hydrogenase. The mutant displayed isms grow rapidly and are found in numerous anaerobic environ- growth equal to wild-type with formate but markedly slower ments. It was recently proposed that methanogens without cyto- growth with hydrogen. The results support the model of electron chromes use flavin-based electron bifurcation from Hdr to simul- bifurcation and suggest that formate, like H2, is closely integrated taneously reduce CoM-S-S-CoB and reduce ferredoxin for Fwd to into the methanogenic pathway. generate formyl-MFR (1). If this were to occur, then ferredoxin reduction by Eha would not be required and net energy conser- energy conservation | Archaea | formate dehydrogenase | vation would result. formylmethanofuran dehydrogenase | F -nonreducing hydrogenase 420 It was our intention to find protein–protein interactions in- volving Hdr that may indicate if there are potential pathways he biochemical steps in methanogenesis from CO2 are well for energy conservation that have eluded prior characterization Tknown, but the interactions that lead to net energy conser- in methanogens without cytochromes. To this end, we per- vation are not well understood. The steps in the pathway are formed experiments with the hydrogenotrophic methanogen, fi diagrammed in Fig. 1 (1). The rst step involves the reduction Methanococcus maripaludis. M. maripaludis is ideal for such an of CO2 and covalent attachment to a unique cofactor, meth- undertaking because of its rapid growth under laboratory con- anofuran (MFR), via the action of formylmethanofuran de- ditions, a well-developed set of genetic tools (3, 4), the ability to hydrogenase (Fwd) to generate formyl-MFR. This represents an grow in continuous culture under conditions of defined nutrient energy-consuming step in the pathway and is dependent on re- limitation (5), and the availability of an exhaustive dataset from duced ferredoxin, thought to be produced at the expense of quantitative measurements of the proteome (6, 7). a chemiosmotic membrane potential via the energy-conserving hydrogenase, Eha. Next, the formyl group is transferred to an- other carrier, tetrahydromethanopterin (H4MPT), and is then Author contributions: K.C.C., T.J.L., J.A.D., I.S., M.H., and J.A.L. designed research; K.C.C., reduced to generate methyl-H4MPT. The methyl group is then P.M.W., T.W., T.J.L., J.A.D., I.S., and J.A.B. performed research; K.C.C., J.A.D., I.S., M.H., and transferred to yet another carrier, coenzyme M (HS-CoM), by J.A.L. analyzed data; and K.C.C., M.H., and J.A.L. wrote the paper. methyl-H4MPT-CoM methyltransferase (Mtr) to generate The authors declare no conflict of interest. + methyl-S-CoM. At this point, Na ions are translocated across This article is a PNAS Direct Submission. W.M. is a guest editor invited by the Editorial the cell membrane. The final step involves reduction of the Board. 1 methyl group to CH4 and capture of HS-CoM by coenzyme B Present address: School of Life Sciences, University of Nevada, Las Vegas, NV 89154. (HS-CoB) to form a CoM-S-S-CoB heterodisulfide. To re- 2To whom correspondence should be addressed. E-mail: [email protected]. generate HS-CoM and HS-CoB, another enzyme is used, het- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. erodisulfide reductase (Hdr). 1073/pnas.1003653107/-/DCSupplemental. 11050–11055 | PNAS | June 15, 2010 | vol. 107 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1003653107 Downloaded by guest on September 23, 2021 CO2 For protein preparations, three experimental strains were Eha Fwd MFR Fd(ox) grown: MM1263 containing His-tagged HdrB1, MM1264 con- Fd(red) H2 + taining His-tagged HdrB2, and MM1265 containing His-tagged membrane Fd(ox) Fd(red) potential FdhA1. Two control strains were also grown: the parental strain Formyl-MFR MM901 containing no His-tagged proteins, and strain MM1262 H4MPT containing the deleted fdh2 and no His-tagged proteins. All five MFR strains were grown under three conditions: hydrogen excess or limitation in a chemostat and batch culture with formate as the Formyl-H4MPT H+ sole electron donor. This process was followed because several Fru F H 420 2 genes are regulated in response to hydrogen availability in H2O F420(ox) M. maripaludis H2 (10), and there could be differences in the com- Methenyl-H4MPT position of the Hdr complex in response to different growth H F H conditions. Cell extracts were made from all 15 cultures and Hmd 2 420 2 Mtd fi fi H+ F (ox) + H+ protein puri cations were done anaerobically using Ni-af nity 420 fi Methylene-H MPT columns. Puri ed samples were analyzed by mass spectrometry. 4 Spectral counts (SC) were tabulated for any protein that F H 420 2 ≥ Mer returned 10 SC in any of the three growth conditions with the F (ox) 420 three experimental strains (Table S1). CH3-H4MPT For each protein, SCs were compared. From an initial in- Na+ CO2 Formate spection of the data it appeared that there were two groups: those translocation Mtr HSCoM that consistently had similar SCs in all five strains (background Fdh proteins), and those that had markedly greater SCs in MM1263 - CH3-S-CoM 2e Hdr and MM1264 (the experimental strains) compared with MM901 HSCoB Mcr (the control strain) or in MM1265 (experimental) to MM1262 2e- H2 CoM-S-S-CoB (control). To distinguish clearly between these groups, three pro- CH Vhu teins among the background proteins (reference proteins) were 4 used as the basis for the calculation of normalized SC ratios Fig. 1. The methanogenic pathway with hypothesized roles for the heter- (Methods). The results are presented in Table S2. A protein was odisulfide reductase complex. Electron flow from formate or hydrogen to considered enriched by copurification with the His-tagged protein Hdr may drive the reduction of the CoM-S-S-CoB heterodisulfide, as well as if the normalized SC ratio was greater by at least three SDs than the reduction of ferredoxin via flavin-mediated electron bifurcation as the average ratio for the 13 background proteins, or if more than outlined by Thauer et al. (1). Eha, energy-conserving hydrogenase; Fdh, for- five SCs were detected in the experimental sample and none was mate dehydrogenase; Fru, F420-reducing hydrogenase; Fwd, formyl-MFR de- fi seen in the control. The results are summarized in Table 1. hydrogenase; Hdr, heterodisul de reductase; Hmd, H2-dependent methylene- fi fi H MPT dehydrogenase; Mcr, methyl-CoM reductase; Mer, methylene-H MPT Subunits from ve different proteins copuri ed with both of the 4 4 His-tagged HdrBs and with His-tagged FdhA1; these were Hdr1, reductase; Mtd, F420-dependent methylene-H4MPT dehydrogenase; Mtr, methyl- H4MPT-CoM methyltransferase; Vhu, F420-nonreducing hydrogenase.
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