Redox-coupled proton pumping drives carbon concentration in the photosynthetic complex I
Authors: Ana P Gamiz-Hernandez
Genji Kurisu
Ville R I Kaila
Sandra K Schuller
Jan M Schuller
Patricia Saura
Jacqueline Thiemann
Marc M Nowaczyk
Journal: Nature Communications (2020)
Discipline: Multidisciplinary
Keywords: Photosynthetic organisms; light energy; energy metabolism; reducing power; carbon dioxide
Affiliations: Department of Structural Cell Biology, Max Planck Institute of Biochemistry, 82152, Martinsried, Germany. ., Department of Biochemistry and Biophysics, Stockholm University, SE-106 91, Stockholm, Sweden., Center of Integrated Protein Science at the Department of Chemistry, Technical University of Munich, Garching, Germany., Plant Biochemistry, Faculty of Biology and Biotechnology, Ruhr University Bochum, 44780, Bochum, Germany., Department of Structural Cell Biology, Max Planck Institute of Biochemistry, 82152, Martinsried, Germany., Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan., Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, 560-0043, Japan., Plant Biochemistry, Faculty of Biology and Biotechnology, Ruhr University Bochum, 44780, Bochum, Germany. ., Department of Biochemistry and Biophysics, Stockholm University, SE-106 91, Stockholm, Sweden. ., Center of Integrated Protein Science at the Department of Chemistry, Technical University of Munich, Garching, Germany. .

Abstract:  Photosynthetic organisms capture light energy to drive their energy metabolism, and employ the chemical reducing power to convert carbon dioxide (CO2) into organic molecules. Photorespiration, however, significantly reduces the photosynthetic yields. To survive under low CO2 concentrations, cyanobacteria evolved unique carbon-concentration mechanisms that enhance the efficiency of photosynthetic CO2 fixation, for which the molecular principles have remained unknown. We show here how modular adaptations enabled the cyanobacterial photosynthetic complex I to concentrate CO2 using a redox-driven proton-pumping machinery. Our cryo-electron microscopy structure at 3.2 Å resolution shows a catalytic carbonic anhydrase module that harbours a Zn2+ active site, with connectivity to proton-pumping subunits that are activated by electron transfer from photosystem I. Our findings illustrate molecular principles in the photosynthetic complex I machinery that enabled cyanobacteria to survive in drastically changing CO2 conditions. 

Retracted: No

PubPeer comments:
 *This comment is a summary of a journal club of u201cRedox-coupled proton pumping drives carbon concentration in the photosynthetic complex Iu201d held in the Savage Lab at UC Berkeley on February 5th 2020. The text was written by Avi Flamholz and edited by Thomas Laughlin, Jack Desmarais and David Savage.* Cyanobacteria are extraordinarily concerned with inorganic carbon flows. They use inorganic carbon in the form of CO2 and HCO3- as their primary carbon source and incorporate it into biomass via the Calvin cycle. All cyanobacteria express inorganic carbon pumps, most of which pump HCO3- from the extracellular space into the cytosol at the expense of ATP or an Na+ gradient. Most cyanobacteria also express CO2 uptake systems, which take up CO2 directly. The structure, molecular composition, and biochemical mechanism of these CO2 pumps has remained enigmatic for decades. Based on previous genetics and biochemistry, it was suggested that these systems involve the binding of auxiliary proteins - called Cup proteins for u201cCO2 uptakeu201d or Chp for u201cCO2 hydrationu201d - to the photosynthetic analog of the respiratory NADH dehydrogenase, NDH-1 [1u20133]. Unlike itu2019s respiratory cousin, NDH-1 lacks a NADH dehydrogenase domain and instead uses ferredoxin as an electron donor, coupling the energy of downhill electron flow from ferredoxin to plastoquinone to the pumping of H+ across the thylakoid membrane [4,5]. The primary role of NDH-1 is to catalyze cyclic electron flow around photosystem I, but negative stain EM suggests that a minority of cyanobacterial NDH-1 complexes are bound to Cup proteins [6,7]. Two forms of Cup-bound NDH-1 complexes, called NDH-13 and and NDH-14, mediate inducible and constitutive CO2 uptake in cyanobacteria respectively [1,3]. Schuller et al. Nature Communications 2020 report the first high-resolution structure of a cyanobacterial CO2 uptake system, the inducible NDH-13. This structure is immensely useful in our efforts to understand the molecular mechanisms of CO2 uptake. As previously suggested by negative stain data, NDH-13 is quite similar in structure to NDH-1. The primary difference is that alternative subunits NdhD3 and NdhF3 displace the canonical proton pumping subunits at the far end of the complex and the CupAS proteins bind to NdhF3. Incidentally, these four proteins, CupAS and NdhDF3, are expressed in a single regulon in many beta cyanobacteria. Many hypothesize that the mechanism of CO2 uptake is u201cvectorial hydration of CO2u201d - that is, coupling of the enzymatic hydration of CO2 to HCO3- to some energy source. It is thought that some of the energy of electron transfer is coupled to a carbonic anhydrase (CA) active site [8]. Canonical carbonic anhydrases catalyze the reversible hydration of CO2 to HCO3-, but coupling this reaction to an energy source would permit the accumulation of HCO3- above equilibrium. If NDH-13 performed such a coupling, it would act as a CO2 pump since extracellular CO2 could enter the cell (passively or through a channel) and then be actively converted to HCO3-. It was previously shown that cyanobacterial membrane preparations have CA activity, and this activity is reduced in Cup knockouts, suggesting that NDH-13 might catalyze an energy-coupled carbonic anhydrase reaction [9]. The most significant finding of Schuller et al. is, in our view, the identification of a Zn2+ ion in the structure near the interface between CupA and NdhF3, forming a potential CA activite site. Nearly all CAs depend on Zn2+ for catalysis. Zn2+ is typically coordinated by three amino acid side chains (typically histidines and cysteines), but here it is coordinated by two side chains (arginine and histidine) and a putative ordered water molecule. Schuller et al. rightly highlight this oddity and run extensive simulations to verify that such an arrangement can, in principle, catalyze the CA reaction. They also perform simulations to identify paths of proton flow through antiporter-like subunits of the complex and identify a channel of non-polar residues in the terminal subunit NdhF3 that might enable flow CO2 through the complex into the CA active site. Presumably HCO3- is disfavored from entering this nonpolar channel, but it is unclear whether the active site Zn2+ is accessible to solvent on the cytoplasmic face of the complex. Since H2O is a reactant in the CA mechanism, it must access the active site at some point in the catalytic cycle, which raises the question of how the reverse reaction (with HCO3- as the substrate) can be excluded when HCO3- has a very high cytosolic concentration in cyanobacteria ( 10 mM). In our view, this valuable paper is vulnerable to two critiques. First, the title and abstract overstate the claims in the text. Second, the paper fails to make clear and testable claims about the mechanism of energy coupling to CO2 hydration at the molecular level. The title, u201cRedox-coupled proton pumping drives carbon concentration in the photosynthetic complex I,u201d is not strongly supported by a paper reporting a structure and simulations. Moreover, none of the simulations reported in the main text touch directly on the means of coupling of electron flow to CO2 uptake, though this is the central claim of the title and abstract. This small critique notwithstanding, previous data from Price, Ogawa, and others do lend credence to the title. What mechanisms might couple oxidation of ferredoxin to CO2 hydration? The authors highlight the presence of a chain of charged residues that starts adjacent to the plastoquinone binding site and terminates near the Zn2+ ion nearly 20 nm away. Based on simulations, they also describe a putative CO2 channel through NdhF3 that also terminates near the Zn2+. These suggestive features could imply that the oxidation state of the plastoquinone is coupled to structural features of the active site (which lies at the far end of the complex) through electrostatic effects. The implication being that some coupling between plastoquinone redox state and the putative CO2 channel could drive unidirectional CO2 hydration, but how exactly? A detailed proposal is not offered. Another model found in the literature, the u201calkaline pocketu201d model, argues that H+ pumping creates a locally basic pH that promotes the hydration of CO2 to HCO3- and H+ [10]. This postulation of an alkaline pocket model is challenging to square with the rate of H+ diffusion, which is faster even than the diffusion of water in water (see Grotthuss mechanism) and makes local pH disequilibrium in bulk water very unlikely. A more proper molecular statement of this model would be that the H+ pumping cycle generates an extremely strong base (a u201cproton holeu201d) near the active site, which drives the CO2 hydration by directly accepting the H+ produced during that reaction [8]. Figure 4 of Schuller et al. diagrams both of the above models at a high-level of abstraction, seeming to propose that both mechanisms collaborate to produce coupling. However, neither the text nor the figures describe either model in enough detail to propose even fanciful experiments that would test or discriminate between them. In either case, some microscopic means of coupling is required. For the CO2 channel - how are electrostatic effects coupled to the putative CO2 channel? Schuller et al. suggest that the CO2 channel opens and closes in a cycle controlled by the oxidation state of plastoquinone, but this does not by itself enable coupling (at least so far as we understand). Moreover, if, as drawn, the active site is open to solvent, how could NDH-13 disfavor the reverse reaction where HCO3- enters and is dehydrated to CO2? For the u201cmolecular alkaline pocketu201d model - how is H+ production during hydration coupled to proton flow? Detailed knowledge of the structure could greatly inform this question, but the authors do not comment on it. We greatly value the efforts of Schuller et al. to understand CO2 uptake mechanisms. Moreover, we recognize that their work touches on one of the most fundamental questions in biochemistry: how do the motions of protein enzymes couple favorable reactions to unfavorable ones? This question is greatly sharpened for large protein complexes like NDH-1, which couple reactions over distances of nearly 20 nm. However, we wish they had more clearly articulated molecular-scale mechanisms that could, even in principle, produce such a coupling through the protein complex. Such an articulation is crucial for imagining experiments probing fundamental questions, so it would be useful if Schuller et al. offered more guidance. These critiques notwithstanding, the structure and simulations reported by Schuller et al. will greatly inform future studies of CO2 uptake in cyanobacteria and other autotrophic lineages. **References** [1]tM. Shibata, H. Ohkawa, T. Kaneko, H. Fukuzawa, S. Tabata, A. Kaplan, T. Ogawa, Distinct constitutive and low-CO2-induced CO2 uptake systems in cyanobacteria: genes involved and their phylogenetic relationship with homologous genes in other organisms, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 11789u201311794. [2]tS.-I. Maeda, M.R. Badger, G. Dean Price, Novel gene products associated with NdhD3 D4-containing NDH-1 complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp. PCC7942, Molecular Microbiology. 43 (2002) 425u2013435. https: doi.org 10.1046 j.1365-2958.2002.02753.x. [3]tB. Klughammer, D. Su00fcltemeyer, M.R. Badger, G.D. Price, The involvement of NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in Synechococcus sp. PCC7002 gives evidence for multiple NDH-1 complexes with specific roles in cyanobacteria, Mol. Microbiol. 32 (1999) 1305u20131315. [4]tJ.M. Schuller, J.A. Birrell, H. Tanaka, T. Konuma, H. Wulfhorst, N. Cox, S.K. Schuller, J. Thiemann, W. Lubitz, P. Su00e9tif, T. Ikegami, B.D. Engel, G. Kurisu, M.M. Nowaczyk, Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer, Science. 363 (2019) 257u2013260. [5]tX. Pan, D. Cao, F. Xie, F. Xu, X. Su, H. Mi, X. Zhang, M. Li, Structural basis for electron transport mechanism of complex I-like photosynthetic NAD(P)H dehydrogenase, Nat. Commun. 11 (2020) 610. [6]tI.M. Folea, P. Zhang, M.M. Nowaczyk, T. Ogawa, E.-M. Aro, E.J. Boekema, Single particle analysis of thylakoid proteins from Thermosynechococcus elongatus and Synechocystis 6803: localization of the CupA subunit of NDH-1, FEBS Lett. 582 (2008) 249u2013254. [7]tM. Birungi, M. Folea, N. Battchikova, M. Xu, H. Mi, T. Ogawa, E.-M. Aro, E.J. Boekema, Possibilities of subunit localization with fluorescent protein tags and electron microscopy examplified by a cyanobacterial NDH-1 study, Biochim. Biophys. Acta. 1797 (2010) 1681u20131686. [8]tG.D. Price, G. Dean Price, S.-I. Maeda, T. Omata, M.R. Badger, Modes of active inorganic carbon uptake in the cyanobacterium, Synechococcus sp. PCC7942, Functional Plant Biology. 29 (2002) 131. https: doi.org 10.1071 pp01229. [9]tX. Han, N. Sun, M. Xu, H. Mi, Co-ordination of NDH and Cup proteins in CO2 uptake in cyanobacterium Synechocystis sp. PCC 6803, Journal of Experimental Botany. 68 (2017) 3869u20133877. https: doi.org 10.1093 jxb erx129. [10]tA. Kaplan, L. Reinhold, CO2CONCENTRATING MECHANISMS IN PHOTOSYNTHETIC MICROORGANISMS, Annual Review of Plant Physiology and Plant Molecular Biology. 50 (1999) 539u2013570. https: doi.org 10.1146 annurev.arplant.50.1.539. ,