Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other sho... more Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other short-chain alkanes, methyl-coenzyme M reductase or Mcr and its homologs play a key role in the global carbon cycle. The McrA subunit possesses up to five conserved post-translational modifications (PTM) at its active site. It was previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To systematically examine its physiological role, mmp10 (locus tag MMP1554), the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool resulting in the complete loss of the 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit. When the deletion mutant Δmmp10 was complemented with the wild-type gene expressed by either a strong or weak promoter, the methylation was fully restored. Compared to the parental strain, the maximal rates of methane formation by whole cells were reduced by 40-60 % in the Δmmp10. The red...
Methyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological... more Methyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological methanogenesis. To better understand its assembly, the recombinant MCR from the thermophile(rMCR) was expressed in the mesophileThe rMCRwas posttranslationally modified correctly and contained McrD and the unique nickel tetrapyrrole coenzyme FSubunits of the native(MCR) were largely absent, suggesting that the recombinant enzyme was formed by an assembly of cotranscribed subunits. Strong support for this hypothesis was obtained by expressing a chimeric operon comprising the His-taggedfromand thefrominThe His-tagged purified rMCR then contained theMcrA and theMcrBDG. The present study prompted us to form a working model for MCR assembly, which can be further tested by the heterologous expression system established here.Approximately 1.6% of the net primary production of plants, algae, and cyanobacteria are processed by biological methane production in anoxic environments. This accounts f...
Journal of the American Chemical Society Jacs, 2005
Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with c... more Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB in methanogenic archaea. Here we present a pulse EPR study of the "ready" form MCR(ox1), providing a detailed description of the spin density and the coordination of coenzyme M (CoM) to the Ni cofactor F430. To achieve this, MCR was purified from cells grown in a 61Ni enriched medium and samples were prepared in D2O with the substrate analogue CoM either deuterated in the beta-position or with 33S in the thiol group. To obtain the magnetic parameters ENDOR and HYSCORE measurements were done at X- and Q-band, and CW EPR, at X- and W-band. The hyperfine couplings of the beta-protons of CoM indicate that the nickel to beta-proton distances in MCR(ox1) are very similar to those in Ni(II)-MCR(ox1-silent), and thus the position of CoM relative to F430 is very similar in both species. Our thiolate sulfur and nickel EPR data prove a Ni-S coordination, with an unpaired spin density on the sulfur of 7 +/- 3%. These results highlight the redox-active or noninnocent nature of the sulfur ligand on the oxidation state. Assuming that MCR(ox1) is oxidized relative to the Ni(II) species, the complex is formally best described as a Ni(III) (d7) thiolate in resonance with a thiyl radical/high-spin Ni(II) complex, Ni(III)-(-)SR <--> Ni(II)-*SR.
Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (Co... more Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (CoB-SH) by the reversible reduction of the heterodisulfide, CoM-S-S-CoB. This reaction recycles the two thiol coenzymes involved in the final step of microbial methanogenesis. Electron paramagnetic resonance (EPR) and variable-temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM-SH revealed a S = 1/2 [4Fe-4S] 3+ cluster, the EPR spectrum of which is broadened in the presence of CoM-33 SH [
Proceedings of the National Academy of Sciences, 2016
Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to... more Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663–10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rum...
Methyl-coenzyme M reductase (MCR) catalyzes the ®nal reaction of the energy conserving pathway of... more Methyl-coenzyme M reductase (MCR) catalyzes the ®nal reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the hetero-disul®de CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F 430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisul®de CoM-S-S-CoB (MCR-silent) were now re®ned at 1.16 A Ê and 1.8 A Ê resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F 430 , of the active site residues and of the modi®ed amino acid residues. Moreover, the observation of 18 Mg 2 and 9 Na ions at the protein surface of the 300 kDa enzyme speci®es typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A Ê resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased¯exibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced speci®c conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.
Heterodisul¢de reductase (Hdr) from methanogenic Archaea catalyzes the reversible reduction of th... more Heterodisul¢de reductase (Hdr) from methanogenic Archaea catalyzes the reversible reduction of the heterodisul¢de (CoM-S^S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Upon reaction of the oxidized enzyme with CoM-SH a unique paramagnetic species is formed, which has been shown to be due to a novel type of [4Fe^4S] 3+ cluster. In this work, it was addressed whether CoM-SH is directly attached to this [4Fe^4S] cluster using CoM-33 SH as substrate and puri¢ed Hdr from Methanothermobacter marburgensis and Methanosarcina barkeri. With both enzymes treatment with CoM-33 SH in the presence of duroquinone as an oxidant resulted in a signi¢cant broadening of the electron paramagnetic resonance spectrum as compared to CoM-SH as substrate. The signal broadening resulted from an unresolved anisotropic hyper¢ne coupling between the 33 S nucleus and the paramagnetic center. The results provide compelling evidence for a direct binding of CoM-SH to the [4Fe^4S] cluster in the active site of the enzyme.
European journal of biochemistry / FEBS, Jan 15, 1997
The selenium-containing F420-reducing hydrogenase from Methanococcus voltae was anaerobically pur... more The selenium-containing F420-reducing hydrogenase from Methanococcus voltae was anaerobically purified to a specific hydrogen-uptake activity of 350 U/mg protein as determined with the natural electron acceptor. The concentrated enzyme was used for EPR-spectroscopic investigations. As isolated, the enzyme showed an EPR spectrum with g(xyz) values of 2.21, 2.15 and 2.01. Illumination of such samples at low temperatures led to an EPR spectrum with g(xyz) values of 2.05, 2.11 and 2.29. These spectra are typical for [NiFe]hydrogenases in the active state. Spectra of samples enriched in 77Se showed a hyperfine interaction between the unpaired spin of the nickel ion and the nuclear spin of one 77Se atom before and after illumination. A 90 degree flip of the electronic z-axis is proposed to explain the hyperfine interaction in both states. This has been demonstrated previously only for the F420-non-reducing hydrogenase from M. voltae, where the selenium atom is present as a selenocysteine ...
Journal of the American Chemical Society, Jan 19, 2015
Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe2... more Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe2S2] ferredoxin (CpFd) provide the only known examples of valence-delocalized [Fe2S2]+ clusters, which constitute a fundamental building block of all higher nuclearity Fe-S clusters. In this work, we have revisited earlier work on the CpFd variants and carried out redox and spectroscopic studies on the [Fe2S2]2+,+ centers in wild-type and equivalent variants of the highly homologous and structurally characterized Aquifex aeolicus ferredoxin 4 (AaeFd4) using EPR, UV-visible-NIR absorption, CD and variable-temperature MCD, and protein-film electrochemistry. The results indicate that the [Fe2S2]+ centers in the equivalent AaeFd4 and CpFd variants reversibly interconvert between similar valence-localized S = 1/2 and valence-delocalized S = 9/2 forms as a function of pH, with pKa values in the range 8.3-9.0, due to protonation of the coordinated serinate residue. However, freezing high-pH samp...
Methyl-coenzyme M reductase catalyzes the reversible synthesis of methane from methyl-coenzyme M ... more Methyl-coenzyme M reductase catalyzes the reversible synthesis of methane from methyl-coenzyme M in methanogenic and ANME-1 and ANME-2 Archaea. The purification procedure for methyl-coenzyme M reductase from Methanothermobacter marburgensis is described. The procedure is an accumulation of almost 30 years of research on MCR starting with the first purification described by Ellefson and Wolfe (Ellefson, W.L., and Wolfe, R.S. (1981). Component C of the methylreductase system of Methanobacterium. J. Biol. Chem.256, 4259-4262). To provide a context for this procedure, some background information is provided, including a description of whole cell experiments that provided much of our knowledge of the behavior and properties of methyl-coenzyme M reductase.
We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identif... more We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N-and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as ؉125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.
Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other sho... more Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other short-chain alkanes, methyl-coenzyme M reductase or Mcr and its homologs play a key role in the global carbon cycle. The McrA subunit possesses up to five conserved post-translational modifications (PTM) at its active site. It was previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To systematically examine its physiological role, mmp10 (locus tag MMP1554), the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool resulting in the complete loss of the 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit. When the deletion mutant Δmmp10 was complemented with the wild-type gene expressed by either a strong or weak promoter, the methylation was fully restored. Compared to the parental strain, the maximal rates of methane formation by whole cells were reduced by 40-60 % in the Δmmp10. The red...
Methyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological... more Methyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological methanogenesis. To better understand its assembly, the recombinant MCR from the thermophile(rMCR) was expressed in the mesophileThe rMCRwas posttranslationally modified correctly and contained McrD and the unique nickel tetrapyrrole coenzyme FSubunits of the native(MCR) were largely absent, suggesting that the recombinant enzyme was formed by an assembly of cotranscribed subunits. Strong support for this hypothesis was obtained by expressing a chimeric operon comprising the His-taggedfromand thefrominThe His-tagged purified rMCR then contained theMcrA and theMcrBDG. The present study prompted us to form a working model for MCR assembly, which can be further tested by the heterologous expression system established here.Approximately 1.6% of the net primary production of plants, algae, and cyanobacteria are processed by biological methane production in anoxic environments. This accounts f...
Journal of the American Chemical Society Jacs, 2005
Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with c... more Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB in methanogenic archaea. Here we present a pulse EPR study of the "ready" form MCR(ox1), providing a detailed description of the spin density and the coordination of coenzyme M (CoM) to the Ni cofactor F430. To achieve this, MCR was purified from cells grown in a 61Ni enriched medium and samples were prepared in D2O with the substrate analogue CoM either deuterated in the beta-position or with 33S in the thiol group. To obtain the magnetic parameters ENDOR and HYSCORE measurements were done at X- and Q-band, and CW EPR, at X- and W-band. The hyperfine couplings of the beta-protons of CoM indicate that the nickel to beta-proton distances in MCR(ox1) are very similar to those in Ni(II)-MCR(ox1-silent), and thus the position of CoM relative to F430 is very similar in both species. Our thiolate sulfur and nickel EPR data prove a Ni-S coordination, with an unpaired spin density on the sulfur of 7 +/- 3%. These results highlight the redox-active or noninnocent nature of the sulfur ligand on the oxidation state. Assuming that MCR(ox1) is oxidized relative to the Ni(II) species, the complex is formally best described as a Ni(III) (d7) thiolate in resonance with a thiyl radical/high-spin Ni(II) complex, Ni(III)-(-)SR <--> Ni(II)-*SR.
Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (Co... more Heterodisulfide reductase (HDR) catalyzes the formation of coenzyme M (CoM-SH) and coenzyme B (CoB-SH) by the reversible reduction of the heterodisulfide, CoM-S-S-CoB. This reaction recycles the two thiol coenzymes involved in the final step of microbial methanogenesis. Electron paramagnetic resonance (EPR) and variable-temperature magnetic circular dichroism spectroscopic experiments on oxidized HDR incubated with CoM-SH revealed a S = 1/2 [4Fe-4S] 3+ cluster, the EPR spectrum of which is broadened in the presence of CoM-33 SH [
Proceedings of the National Academy of Sciences, 2016
Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to... more Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663–10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rum...
Methyl-coenzyme M reductase (MCR) catalyzes the ®nal reaction of the energy conserving pathway of... more Methyl-coenzyme M reductase (MCR) catalyzes the ®nal reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the hetero-disul®de CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F 430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisul®de CoM-S-S-CoB (MCR-silent) were now re®ned at 1.16 A Ê and 1.8 A Ê resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F 430 , of the active site residues and of the modi®ed amino acid residues. Moreover, the observation of 18 Mg 2 and 9 Na ions at the protein surface of the 300 kDa enzyme speci®es typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A Ê resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased¯exibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced speci®c conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.
Heterodisul¢de reductase (Hdr) from methanogenic Archaea catalyzes the reversible reduction of th... more Heterodisul¢de reductase (Hdr) from methanogenic Archaea catalyzes the reversible reduction of the heterodisul¢de (CoM-S^S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Upon reaction of the oxidized enzyme with CoM-SH a unique paramagnetic species is formed, which has been shown to be due to a novel type of [4Fe^4S] 3+ cluster. In this work, it was addressed whether CoM-SH is directly attached to this [4Fe^4S] cluster using CoM-33 SH as substrate and puri¢ed Hdr from Methanothermobacter marburgensis and Methanosarcina barkeri. With both enzymes treatment with CoM-33 SH in the presence of duroquinone as an oxidant resulted in a signi¢cant broadening of the electron paramagnetic resonance spectrum as compared to CoM-SH as substrate. The signal broadening resulted from an unresolved anisotropic hyper¢ne coupling between the 33 S nucleus and the paramagnetic center. The results provide compelling evidence for a direct binding of CoM-SH to the [4Fe^4S] cluster in the active site of the enzyme.
European journal of biochemistry / FEBS, Jan 15, 1997
The selenium-containing F420-reducing hydrogenase from Methanococcus voltae was anaerobically pur... more The selenium-containing F420-reducing hydrogenase from Methanococcus voltae was anaerobically purified to a specific hydrogen-uptake activity of 350 U/mg protein as determined with the natural electron acceptor. The concentrated enzyme was used for EPR-spectroscopic investigations. As isolated, the enzyme showed an EPR spectrum with g(xyz) values of 2.21, 2.15 and 2.01. Illumination of such samples at low temperatures led to an EPR spectrum with g(xyz) values of 2.05, 2.11 and 2.29. These spectra are typical for [NiFe]hydrogenases in the active state. Spectra of samples enriched in 77Se showed a hyperfine interaction between the unpaired spin of the nickel ion and the nuclear spin of one 77Se atom before and after illumination. A 90 degree flip of the electronic z-axis is proposed to explain the hyperfine interaction in both states. This has been demonstrated previously only for the F420-non-reducing hydrogenase from M. voltae, where the selenium atom is present as a selenocysteine ...
Journal of the American Chemical Society, Jan 19, 2015
Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe2... more Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe2S2] ferredoxin (CpFd) provide the only known examples of valence-delocalized [Fe2S2]+ clusters, which constitute a fundamental building block of all higher nuclearity Fe-S clusters. In this work, we have revisited earlier work on the CpFd variants and carried out redox and spectroscopic studies on the [Fe2S2]2+,+ centers in wild-type and equivalent variants of the highly homologous and structurally characterized Aquifex aeolicus ferredoxin 4 (AaeFd4) using EPR, UV-visible-NIR absorption, CD and variable-temperature MCD, and protein-film electrochemistry. The results indicate that the [Fe2S2]+ centers in the equivalent AaeFd4 and CpFd variants reversibly interconvert between similar valence-localized S = 1/2 and valence-delocalized S = 9/2 forms as a function of pH, with pKa values in the range 8.3-9.0, due to protonation of the coordinated serinate residue. However, freezing high-pH samp...
Methyl-coenzyme M reductase catalyzes the reversible synthesis of methane from methyl-coenzyme M ... more Methyl-coenzyme M reductase catalyzes the reversible synthesis of methane from methyl-coenzyme M in methanogenic and ANME-1 and ANME-2 Archaea. The purification procedure for methyl-coenzyme M reductase from Methanothermobacter marburgensis is described. The procedure is an accumulation of almost 30 years of research on MCR starting with the first purification described by Ellefson and Wolfe (Ellefson, W.L., and Wolfe, R.S. (1981). Component C of the methylreductase system of Methanobacterium. J. Biol. Chem.256, 4259-4262). To provide a context for this procedure, some background information is provided, including a description of whole cell experiments that provided much of our knowledge of the behavior and properties of methyl-coenzyme M reductase.
We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identif... more We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N-and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as ؉125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.
Uploads
Papers by Evert Duin