Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria

Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria Aadi Prabhu, William Cramer This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3463667/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A working concept for the regulation of electron transport in oxygenic photosynthesis is that the electron transfer rate between the two photosystems, PSI and PSII, is governed by a ‘Q-cycle’ pathway operating in the electron transport chain which connects the two ‘reaction center’ complexes. The ‘Q-cycle’ concept was initially inferred from studies on mitochondrial electron transport. This concept has been assumed to be relevant to the electron transport pathways operating in oxygenic photosynthesis, with the majority of studies done on chloroplasts or thylakoid membranes The present study examines the existence and properties of a putative ‘Q-cycle’ in cyanobacteria. Light-induced spectral changes associated with cytochrome redox reactions in intact cells of the cyanobacterium Synechococcus sp . corresponded to the oxidation-reduction of cytochrome f . A correlated reduction of heme b 6 was, however, not observed. The absence of significant cytochrome b reduction might be considered inconsistent with the set of electron transfer events associated conceptually with a ‘Q-cycle’ model of the electron transfer events in the chain. However, because heme b 6 in the intact cyanobacteria is mostly reduced, it is not observable as a net electron acceptor of the plastoquinol or semiquinone formed by electron transfer from photosystem II. The redox environment of intact cyanobacteria in the dark resting state has an ambient potential sufficiently reducing that the ‘Q-cycle’ pathway for electron transport, well studied and characterized for function in isolated thylakoid membranes or chloroplasts, is not observed. This apparent quandary’ is a consequence of the reducing (negative potential) intracellular redox environment of cyanobacteria, which imposes a reduced state on the b -hemes, thereby preventing observation of their light-induced reduction. Figures Figure 1 Figure 2 Figure 3 Introduction Under conditions of optimum light intensity and carbon dioxide concentration, the rate-determining step of oxygenic photosynthesis via non-cyclic electron transport is electron transfer from plastoquinol (PQH 2 )/semiquinone) to the high potential non-heme iron-sulfur (‘Rieske’) protein and a low potential cytochrome (cyt b 6 ) heme of the electron transport chain. It has been proposed that details of electron transfer in the quinol oxidation reaction are described by a ‘Q-cycle’ model, based on the realization of the ubiquinone fluidity proposed for the ubiquinol-cytochrome b reaction in mitochondria ( Mitchell, 1976; Slater, 1983; Schneider et al., 1985; Trumpower, 2002 ), and subsequently studied in photosynthetic bacteria ( Crofts and Meinhardt, 1983; Crofts et al ., 1983; Joliot, P. and Joliot, A., 2002 ), its properties described in some detail in a textbook Cramer and Knaff ( 1990 ). Regarding application of the Q-cycle model to oxygenic photosynthesis, a goal of the present study is to provide such documentation for the redox changes associated with quinol oxidation, and the cytochrome redox changes, in a cyanobacterium (PC7002, Synechococcus sp ). A feature of the present study is a comparison of these redox properties with those reported previously for plant thylakoid membranes ( Cramer and Butler, 1967; Cramer and Hasan, 2016 ). It is noted in the present discussion that the two trans-membrane b hemes, one on the electrochemically negative (n) and one on the positive (p), ‘stromal’ and ‘granal’ sides, respectively) in the cytochrome b 6 f complex, with standard potentials -0.1 - + 0.1 V) are mostly reduced in the active growth phase of cultures of the cyanobacterium, thus defining a redox environment seemingly unfavorable for operation of a Q cycle. It was, therefore, inferred that proton translocation associated with quinol oxidation at this site might occur through a different (e. g., H + pump) mechanism, involving the high potential iron-sulfur (Rieske) protein ( Kramer and Crofts, 1990; Papa et al ., 1983). Here it is proposed that the different results obtained in ‘flash-kinetic’ experiments with (i) isolated plant chloroplasts compared to (ii) intact photosynthetic microbial cells do not imply any difference in the pathways and mechanisms of oxygenic photosynthetic electron transport, but rather a difference in the ambient redox environment of the hydrophobically intact cell interior and the more polar environment of the redox carriers in isolated chloroplasts. Materials and Methods 1. Growth of Cyanobacterial Cultures . Synechococcus sp. PCC 7002, wild-type, were grown in medium A ( Hasan et al ., 2018 ) under optimal growth conditions, 38°C with an incident light intensity of 100–150 micro Einsteins·m −2 ·s −1 in 2% (v/v) CO 2 /air. The cell titer was assayed using a Cary 3 spectrophotometer through the optical density of the cell culture measured at 660 nm relative to that at a reference wavelength of 730 nm, using a spectrophotometer with the light collection and detector geometry designed for a high efficiency of collection of light scattered from the turbid cell suspension. 2, Preparation of Spinach Chloroplast Thylakoid Membranes . Thylakoid membranes were prepared according to ( Hasan and Cramer (2014); Hasan et al., 2014). Spinach leaves (15 g) were homogenized (in the presence of 100 ml of buffer A or grinding buffer at high speed for 30 seconds. The resulting mixture was passed through a cloth filter. The filtrate was then separated by centrifugation (2 min, 2,000 x g, 4 o C), and the isolated pellet resuspended in ‘buffer B’ ( Cramer and Hasan, 2016 ) and incubated on ice for 10 min. The solution was centrifuged twice at 4000 rpm for 10 min, and the resulting sediment stored in buffer B containing protease inhibitors. Aliquots of this suspension were used to carry out determinations of chlorophyllconcentration. 3 . Flash Kinetic Spectroscopy . To measure cytochrome f oxidation, wild type Synechococcus was used at a final chlorophyll concentration of 50-100 μg/ml in the presence of 0.5 mM methyl viologen (MV) as the terminal electron acceptor. Flash-induced oxidation of cytochrome f , using a triggered Xenon flash single-beam spectrometer ( Zhang et al., 2008 ) was measured at 554 nm relative to 540 nm as a reference wavelength, thus generating the difference absorbance signal, ΔA 554–540 . An ‘action spectrum’ which describes the light-induced oxidation of cytochrome f was obtained by repeating this measurement over the wavelength range (548–560 nm) that spans the cytochrome alpha-band spectral domain. The same procedure was used to obtain an action spectrum associated with the light-induced redox change for heme b 6 reduction in spinach thylakoid membranes, with the peak of its chemical redox difference spectrum at 563 nm, using a reference wavelength of 575 nm. Duroquinol at a final concentration of 0.5 mM, and the ‘uncoupler’ gramicidin (5 μM) were added to the reaction mixture to facilitate regeneration of reduced plastoquinone and cytochrome f . The cell suspension was diluted to an optical density of 1.0 at 730 nm for spectrophotometric analysis, the output signal referenced to the pre-flash output function, and a noise suppression algorithm utilized to minimize noise in the signal and detector output. Results The pattern of absorbance and resulting spectral changes associated with light- and chemically - induced changes in the oxidation state of cytochrome f has been documented in previous studies on spinach thylakoids and C. reinhardtii ( Cramer and Hasan, 2016 ). The present study extended this analysis to the redox changes of the cytochrome b 6 f complex in the cyanobacterium, Synechococcus sp. PCC 7002. The light-induced redox changes were referenced to those obtained by actinic Illumination of spinach chloroplasts, suspended at a concentration of 50-100 μg/ml chlorophyll with methyl viologen (0.5 mM), as the electron acceptor. The peak of the spectrum generated to characterize the extent of the cytochrome-specific redox changes ( Cramer and Hasan, 2016 ; Kramer and Crofts, 1990 ) is close to 554 nm ( Fig. 1A ), the α- band (in green region of spectrum)-peak of the cytochrome f redox difference spectrum. A spectrum of the light-induced absorbance change, an example shown in Fig. 1B , was generated from the set of individual absorbance measurements at ( Fig. 1A ). The peak of the difference spectrum as defined by ‘best fit’ analysis shows that the spectrum peaks at 555 nm. The difference spectrum for oxidation of heme b in the b 6 f complex was centered at 563-564 nm ( Fig. 1B) . These spectra are consistent with the well-documented precedent for the spectrum for light-induced oxidation of cytochrome f ( Cramer and Hasan, 2016; Hasan et al ., 2018 ), and also provide a spectrophotometric reference for the additional studies reported here on the amplitude and kinetics of the light-induced redox turnover of b -cytochromes in intact cyanobacteria. The amplitude and time course of ‘dark reduction,’ the redox change in the dark to light-induced oxidation of cytochrome f in spinach thylakoid membranes, measured by the absorbance change at 554 nm relative to a reference change measured at 540 nm, is shown ( Fig. 2A). The first 40 ms in the display show a baseline which displays ( i ) the background noise in the measurement followed by ( ii ) a spike arising from leak into the detector of the light flash at approximately 40 ms into the recording In the absence or presence of a sample. The amplitude of the effective absorbance change associated with a leak of the relatively intense actinic light flash, if displayed alone, would decrease immediately after termination of the light flash. However, a relatively slow decrease in absorbance is seen with a half-life of 8-10 msec which arises from the absorbance change associated with (i) the light-induced oxidation, the detailed time course not resolved here, and (ii) the subsequent ‘dark reduction’ of cytochrome f after termination of the light flash. The time course of the initial light-induced oxidation of the cytochrome was not resolved in these measurements. Under the same experimental conditions, similar measurements were done at a measuring light wavelength of 563 nm to determine the spectral properties of the heme b 6 redox change ( Fig. 2B ). The reaction medium contained duroquinol as a reductant of the quinone pool and gramicidin as an uncoupler of electron transfer. A ‘slow’ oxidation of heme b , consistent with prediction and previous observations was observed. Presumably because of the difference in rateconstants for oxidation and reduction, the change in absorbance for heme b 6 is significantly smaller than that of cytochrome f . The critical experiment which describes the presence of these absorbance changes in intact cells, spectrophotometric measurement at the alpha-band absorbance peaks associated with cytochromes f and b 6 , is shown ( Figs. 3A, B) . Materials and Methods 1. Growth of Cyanobacterial Cultures . Synechococcus sp. PCC 7002, wild-type, were grown in medium A ( Hasan et al ., 2018 ) under optimal growth conditions, 38°C with an incident light intensity of 100–150 micro Einsteins·m − 2 ·s − 1 in 2% (v/v) CO 2 /air. The cell titer was assayed using a Cary 3 spectrophotometer through the optical density of the cell culture measured at 660 nm relative to that at a reference wavelength of 730 nm, using a spectrophotometer with the light collection and detector geometry designed for a high efficiency of collection of light scattered from the turbid cell suspension. 2 , Preparation of Spinach Chloroplast Thylakoid Membranes . Thylakoid membranes were prepared according to ( Hasan and Cramer (2014); Hasan et al., 2014). Spinach leaves (15 g) were homogenized (in the presence of 100 ml of buffer A or grinding buffer at high speed for 30 seconds. The resulting mixture was passed through a cloth filter. The filtrate was then separated by centrifugation (2 min, 2,000 x g, 4 o C), and the isolated pellet resuspended in ‘buffer B’ ( Cramer and Hasan, 2016 ) and incubated on ice for 10 min. The solution was centrifuged twice at 4000 rpm for 10 min, and the resulting sediment stored in buffer B containing protease inhibitors. Aliquots of this suspension were used to carry out determinations of chlorophyllconcentration. 3 . Flash Kinetic Spectroscopy . To measure cytochrome f oxidation, wild type Synechococcus was used at a final chlorophyll concentration of 50–100 µg/ml in the presence of 0.5 mM methyl viologen (MV) as the terminal electron acceptor. Flash-induced oxidation of cytochrome f , using a triggered Xenon flash single-beam spectrometer ( Zhang et al. , 2008 ) was measured at 554 nm relative to 540 nm as a reference wavelength, thus generating the difference absorbance signal, ΔA 554–540 . An ‘action spectrum’ which describes the light-induced oxidation of cytochrome f was obtained by repeating this measurement over the wavelength range (548–560 nm) that spans the cytochrome alpha-band spectral domain. The same procedure was used to obtain an action spectrum associated with the light-induced redox change for heme b 6 reduction in spinach thyla- Discussion It is shown here that a major difference exists between the results of visible light flash excitation experiments on the b -hemes obtained ( a ) with thylakoid (e. g., spinach) membranes and ( b ) cyanobacteria, represented here by the studies on Synechococcus ( Fig. 2B and 3B, respectively ) . Thus, based on a measured absorbance change of approximately 1.5 x 10 -3 for cytochrome f at 554 nm ( Fig. 3A ), a significant amplitude for photoreduction of heme b 6 , assayed through the magnitude of the absorbance difference at 563-564 nm, was not detected. The detector of the spectrophotometric device has an intrinsic noise level of 0.5 x 10 -4 absorbance units. Thus, if there is a light-induced change in the b 6 heme, it is substantially smaller than would be expected for any significant amplitude of reduction. In summary, the ratio of the light-induced absorbance change between heme b 6 and cytochrome f is significantly smaller than 0.5, partly a consequence of the structural proximity of cytochrome f to the photosystem I reaction center. These studies suggest an alternate pathway and mechanism for energy transduction linked to electron transport in the ‘main’ electron transport chain linking the two photosystems. Regarding the location of this additional site for energy transduction, It has been proposed that a significant component of the proton electrochemical potential gradient in oxygenic photosynthesis could be provided through a pathway and mechanism of proton translocation involving conformation changes of the Rieske protein present in the mitochondrial cytochrome bc 1 complex ( Papa et al ., 1983). The latter model combines ‘proton-motive’ catalysis by quinone bound to the cytochrome bc 1 complex, and proton conduction along intra-membrane, trans-membrane pathways in the apoproteins of the cytochrome bc 1 complex. Declarations Acknowledgments WACproposedthe study; AP carried out the spectrophotometric analysis as part of an undergraduate research project. The authorsthank the College of Science. Purdue University, for providing a summer research Fellowship to AP, and the Department of Biological Sciences, for providing technical support of the facilities, with no competing interests. We thank Ms. Rebecca Harding for assistance in the assembly and submission of the manuscript. References Mitchell, P. (1976) Possible Molecular Mechanisms of the Proton Motive Function of Cytochrome Systems. J. Theor. Biol, 62, 327–367. E. C. Slater (1983) The ‘Q cycle,’ A Ubiquitous Mechanism of Electron Transfer. Trends Biochem. Sci., 8, 239–242. Schneider, H., J. J. Lemasters, and C. R. Hackenbrock (1985) Membrane fluidity and mobility of ubiquinone. In Coenzyme Q (ed. (G. Lenaz, ed.), pp. 201–204. John Wiley, New York. Trumpower, B. L. (2002) A concerted alternating sites mechanism of ubiquinol oxidation by the cytochrome bc (1) complex. Biochim Biophys Acta, 1555, 166–173. Crofts, A. R., S. W. Meinhardt (1983) The role of the quinone pool A modified Q-cycle mechanism for the cyclic electron-transfer chain of Rhodopseudomonas sphaeroides . Crofts, A. R. S. W. Meinhardt, and K. B. Jones (1983) The role of the quinone pool in the cyclic electron transfer chain of Rhodopseudomonas sphaeroides : a modified Q cycle mechanism. Biochim Biophys Acta, 723, 202–218. Joliot, P., and A. Joliot (2002) Cyclic electron transfer in plant leaf. Proc. Natl. Acad Sci., U.S , 99, 10209–10214. Cramer, W. A. and D. B. Knaff (1990) ‘ The Quinone Connection , chapt. 5 in Energy Transduction in Biological Membranes , pp. 545, a textbook, Springer-Verlag, ISBN 0-387-96761-3. Cramer, W. A. and W. L. Butler (1967) Light induced absorption changes of two cytochrome b components in the electron transport chain of spinach chloroplasts. Biochim. Biophys. Acta 143, 332–339. Cramer, W. A. and S. Saif Hasan (2016) The Cytochrome b 6 f Lipoprotein Complex ; pp. 177–207 in “ Cytochrome Complexes: Evolution, Structures, Energy Transduction, and Signaling ,” Eds. W. A. Cramer and T. Kallas, volume 41 in the Series " Advances in Photosynthesis and Respiration ,” eds. Govindjee and T. Sharkey" (Springer, Dordrecht).ISSN 1572 – 0233; ISBN 978-94-017-7479-6; DOI 10.1007/978-94-017-7481-9 . Kramer, D. M., and A. R. Crofts (1990) A modified Q cycle mechanism for electron transfer in chloroplasts. Current Research in Photosynthesis , Proc. VIII Int. Conf. Photosynthesis , 2189–2192. Papa, S., M. Lorusso, D. Boffoli, and E. Bellowmo (1983) Redox-linked proton translocation in the b–c 1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles: general characteristics and control of electron transport. European Journal of Biochemistry 137, 405–412. Hasan, S. S., D. Baniulis, E. Yamashita, M. V. Zhalnina, S. D. Zakharov, J. T. Stofleth, and W. A. Cramer (2018) Methods for Studying Interactions of Detergents and Lipids with α-Helical and β-Barrel Integral Membrane Proteins. Current Protocols in Protein Science, Novus, J. Wiley and Sons , https://doi.org/10.1002/0471140864.ps2907s74 Hasan, S. S. and W. A. Cramer (2014) Internal lipid architecture of the hetero-oligomeric cytochrome b 6 f lipoprotein complex. Structure , 22, 1008–1015. Hasan S. S., S. D. Zakharov, A. Chauvet, V. Stadnytskyi, S. Savikhin and W. A. Cramer (2014) A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex. J Phys Chem B 118, 6614–6625. Zhang, S., S. Hasan, and W. A. Cramer (2008) Structure - Function of the Cytochrome b6f Complex. Photochemistry and Photobiology, 84, 1349–1358 Cramer, W. A. (2019) Structure-Function of the Cytochrome b 6 f Lipoprotein Complex: A Scientific Odyssey and Personal Perspective. Photosyn Res. 139, 52–65 Hasan S. S., S. D. Zakharov, A. Chauvet, V. Stadnytskyi, S. Savikhin and W. A. Cramer (2014) A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex. J Phys Chem B 118, 6614–6625 Papa, S., M. Lorusso, D. Boffoli, and E. Bellowmo (1983) Redox-linked proton translocation in the b–c 1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. Eur. J. Biochem. 137: 405–412. Baniulis, D., E Yamashita, H Zhang, S Saif Hasan, WA Cramer (2014). Photochemistry and conservation of lipid functions in cytochrome bc complexes. Structure 22, 1008–1015, Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3463667","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":241335180,"identity":"e36b602b-e5bc-4786-bd1f-167007294098","order_by":0,"name":"Aadi Prabhu","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aadi","middleName":"","lastName":"Prabhu","suffix":""},{"id":241335182,"identity":"3ff0c912-b1f5-4edf-9787-ab0c28b5f86d","order_by":1,"name":"William Cramer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYHACxgOMDTYghgGY10CMHqCWNNK1HCZBi8Hxww8O/NxxPs/g9uFtEh8YbGQ3HCCk5UyawcHeM7eLDc6llUnOYEgzJqjF7EAOwwHettuJG87wmEnzMBxOJKzl/BuGg3/bzkG0/GH4T4SWGzkMh3nbDkC0AIOCsBb7G88MDsueSS6WPMNWbNljkGw8k5AWyf7khw/f7rDL4zvDvPHGjwo72T5CWmAgAUIZEKkcScsoGAWjYBSMAiwAAJwFTLSNbv50AAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Cramer","suffix":""}],"badges":[],"createdAt":"2023-10-18 20:44:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3463667/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3463667/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":45166977,"identity":"59ea3cd6-df7d-4267-9850-82bdcd673348","added_by":"auto","created_at":"2023-10-24 17:07:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112380,"visible":true,"origin":"","legend":"\u003cp\u003eAction spectra for light (flash)-induced absorbance changes of spinach thylakoids in the heme spectrophotometric α-band domain.\u003cstrong\u003e \u003c/strong\u003eThe spectrum displays a peak at 554 nm, associated with reduction of cytochrome \u003cem\u003ef\u003c/em\u003e, and\u003cstrong\u003e \u003c/strong\u003ea ‘shoulder’ at 564.5 nm contributed by the reduction of heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e. \u0026nbsp;(\u003cstrong\u003eA\u003c/strong\u003e) (Top) Spectrum for oxidation of cytochrome \u003cem\u003ef\u003c/em\u003e in spinach thylakoid membranes, showing flash-induced absorbance changes relative to those measured at a reference wavelength of 540 nm. A ‘Gaussian’ bell-shaped function was plotted (maximum amplitude, 554 nm, peak, 555 nm) with parameters adjusted for a best fit to the data. (\u003cstrong\u003eB\u003c/strong\u003e) Spectrum for light-induced heme b\u003csub\u003e6\u003c/sub\u003e reduction in spinach thylakoids. The amplitude of the flash-induced absorbance change is plotted as a function of wavelength with respect to a reference absorbance change at 575 nm. A ‘best fit’ function is shown with a peak at 555 nm. (\u003cstrong\u003eC\u003c/strong\u003e) Absorbance spectrum for redox-linked spectral changes spanning the α-band region of cytochromes \u003cem\u003ef \u003c/em\u003eand \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e, obtained with active cyanobacteria.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3463667/v1/f2d6f759b719d26754aea9a9.png"},{"id":45166975,"identity":"19daa5ac-ff4c-45c6-b1ec-75fdb836400a","added_by":"auto","created_at":"2023-10-24 17:07:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111873,"visible":true,"origin":"","legend":"\u003cp\u003eKinetics of (\u003cstrong\u003eA\u003c/strong\u003e) the absorbance change in spinach thylakoids at 554 nm (peak of cytochrome f difference spectrum); noise level Δ = +/- 5 x 10\u003csup\u003e-5\u003c/sup\u003e absorbance units, and (\u003cstrong\u003eB\u003c/strong\u003e) change in absorbance in the heme \u003cem\u003eb\u003c/em\u003e domain of the spectrum \u0026nbsp;as a function of time in the same medium at a wavelength of 563 nm (peak of heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e reduced minus oxidized difference spectrum) minus the change of a reference signal at 575 nm. A negative peak resulting from a leak of the light flash, followed by a slow (multi-millisecond) return to the baseline, the latter kinetic component characteristic of heme b\u003csub\u003e6\u003c/sub\u003e reduction\u003cstrong\u003e. \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003eratio of the amplitude of the absorbance change of cytochrome \u003cem\u003ef \u003c/em\u003eto that of heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6 \u003c/sub\u003eis approximately two.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3463667/v1/af73f3fabd10acc4e1777a00.png"},{"id":45167810,"identity":"529e61a2-98c3-456e-8e2f-d57d52e40f41","added_by":"auto","created_at":"2023-10-24 17:15:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":114172,"visible":true,"origin":"","legend":"\u003cp\u003eFlash-induced spectroscopy indicates that intact cells are normally reduced in resting state. (\u003cstrong\u003eA\u003c/strong\u003e) Flash-induced absorbance change measured in intact cyanobacteria at 554 nm (referenced to the absorbance change at 540 nm). The graph resembles that seen in spinach thylakoid membranes for cytochrome \u003cem\u003ef\u003c/em\u003e (e. g., as in \u003cstrong\u003eCramer and Hasan (2016\u003c/strong\u003e). (\u003cstrong\u003eB\u003c/strong\u003e) Light-induced absorbance change in intact cyanobacteria measured at 563 nm relative to the change at 575 nm (control). No significant absorbance change associated with reduction of heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e is observed.\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3463667/v1/1089271e9c08899baaf43d1e.png"},{"id":77862386,"identity":"097d7d74-3db8-4ea4-9e1b-bd1016016c8a","added_by":"auto","created_at":"2025-03-06 08:54:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1088286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3463667/v1/d075727b-c749-4e45-9c1e-83b7414f8424.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUnder conditions of optimum light intensity and carbon dioxide concentration, the rate-determining step of oxygenic photosynthesis via non-cyclic electron transport is electron transfer from plastoquinol (PQH\u003csub\u003e2\u003c/sub\u003e)/semiquinone) to the high potential non-heme iron-sulfur (\u0026lsquo;Rieske\u0026rsquo;) protein and a low potential cytochrome (cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) heme of the electron transport chain. \u0026nbsp;It has been proposed that details of electron transfer in the quinol oxidation reaction are described by a \u0026lsquo;Q-cycle\u0026rsquo; model, based on the realization of the ubiquinone fluidity proposed for the ubiquinol-cytochrome \u003cem\u003eb\u003c/em\u003e reaction in mitochondria (\u003cstrong\u003eMitchell, 1976; Slater, 1983; Schneider et al., 1985; Trumpower, 2002\u003c/strong\u003e), and subsequently studied in photosynthetic bacteria (\u003cstrong\u003eCrofts and Meinhardt, 1983; Crofts \u003cem\u003eet al\u003c/em\u003e., 1983; Joliot, P. and Joliot, A., 2002\u003c/strong\u003e), its properties described in some detail in a textbook \u003cstrong\u003eCramer and Knaff\u003c/strong\u003e (\u003cstrong\u003e1990\u003c/strong\u003e). Regarding application of the Q-cycle model to oxygenic photosynthesis, a goal of the present study is to provide such documentation for the redox changes associated with quinol oxidation, and the cytochrome redox changes, in a cyanobacterium (PC7002, \u003cem\u003eSynechococcus sp\u003c/em\u003e). A feature of the present study is a comparison of these redox properties with those reported previously for plant thylakoid membranes\u0026nbsp;(\u003cstrong\u003eCramer and Butler, 1967; Cramer and Hasan, 2016\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;It is noted in the present discussion that the two trans-membrane \u003cem\u003eb\u003c/em\u003e hemes, one on the electrochemically negative (n) and one on the positive (p), \u0026lsquo;stromal\u0026rsquo; and \u0026lsquo;granal\u0026rsquo; sides, respectively) in the cytochrome\u003cem\u003e\u0026nbsp;b\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e\u003cem\u003ef\u003c/em\u003e complex, with standard potentials -0.1 - + 0.1 V) are mostly reduced in the active growth phase of cultures of the cyanobacterium, thus defining a redox environment seemingly unfavorable for operation of a Q cycle. It was, therefore, inferred that proton translocation associated with quinol oxidation at this site might occur through a different (e. g., H\u003csup\u003e+\u003c/sup\u003e pump) mechanism, involving the high potential iron-sulfur (Rieske) protein (\u003cstrong\u003eKramer and Crofts, 1990; Papa \u003cem\u003eet al\u003c/em\u003e., 1983).\u003c/strong\u003e\u0026nbsp; Here it is proposed that the different results obtained in \u0026lsquo;flash-kinetic\u0026rsquo; experiments with \u003cstrong\u003e(i)\u003c/strong\u003e isolated plant chloroplasts compared to \u003cstrong\u003e(ii)\u003c/strong\u003e intact photosynthetic microbial cells do not imply any difference in the pathways and mechanisms of oxygenic photosynthetic electron transport, but rather a difference in the ambient redox environment of the hydrophobically intact cell interior and the more polar environment of the redox carriers in isolated chloroplasts.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e1. Growth of Cyanobacterial\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eCultures\u003c/strong\u003e\u003cem\u003e. Synechococcus\u003c/em\u003e \u003cem\u003esp.\u003c/em\u003e PCC 7002, wild-type, were grown in medium A (\u003cstrong\u003eHasan \u003cem\u003eet al\u003c/em\u003e., 2018\u003c/strong\u003e) under optimal growth conditions, 38\u0026deg;C with an incident light intensity of 100\u0026ndash;150 micro Einsteins\u0026middot;m\u003csup\u003e\u0026minus;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;1\u003c/sup\u003e in 2% (v/v) CO\u003csub\u003e2\u003c/sub\u003e/air. The cell titer was assayed using a Cary 3 spectrophotometer through the optical density of the cell culture measured at 660 nm relative to that at a reference wavelength of 730 nm, using a spectrophotometer with the light collection and detector geometry designed for a high efficiency of collection of light scattered from the turbid cell suspension.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2,\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003cstrong\u003ePreparation of Spinach Chloroplast Thylakoid Membranes\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThylakoid membranes were prepared according to (\u003cstrong\u003eHasan and Cramer (2014); Hasan et al., 2014).\u0026nbsp;\u003c/strong\u003e Spinach leaves (15 g) were homogenized (in the presence of 100 ml of buffer A or grinding buffer at high speed for 30 seconds. The resulting mixture was passed through a cloth filter. The filtrate was then separated by centrifugation (2 min, 2,000 x g, 4\u003csup\u003eo\u003c/sup\u003eC), and the isolated pellet resuspended in \u0026lsquo;buffer B\u0026rsquo; (\u003cstrong\u003eCramer and Hasan, 2016\u003c/strong\u003e) and incubated on ice for 10 min. The solution was centrifuged twice at 4000\u0026nbsp;rpm\u0026nbsp;for 10 min, and the resulting sediment stored in buffer B containing protease inhibitors.\u0026nbsp;Aliquots of this suspension were used to carry out determinations of chlorophyllconcentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e. Flash Kinetic Spectroscopy\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eTo measure cytochrome\u003cem\u003e\u0026nbsp;f\u003c/em\u003e oxidation, wild type \u003cem\u003eSynechococcus\u003c/em\u003e was used at a final chlorophyll concentration of 50-100 \u0026mu;g/ml in the presence of 0.5 mM methyl viologen (MV) as the terminal electron acceptor. Flash-induced oxidation of cytochrome \u003cem\u003ef\u003c/em\u003e, using a triggered Xenon flash single-beam spectrometer (\u003cstrong\u003eZhang \u003cem\u003eet al.,\u003c/em\u003e 2008\u003c/strong\u003e)\u0026nbsp;was measured at 554 nm relative to 540 nm as a reference wavelength, thus generating the difference absorbance signal, \u0026Delta;A\u003csub\u003e554\u0026ndash;540\u003c/sub\u003e. An \u0026lsquo;action spectrum\u0026rsquo; which describes the light-induced oxidation of cytochrome \u003cem\u003ef\u003c/em\u003e was obtained by repeating this measurement over the wavelength range (548\u0026ndash;560 nm) that spans the cytochrome alpha-band spectral domain. The same procedure was used to obtain an action spectrum associated with the light-induced redox change for heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e reduction in spinach thylakoid membranes, with the peak of its chemical redox difference spectrum at 563 nm, using a reference wavelength of 575 nm. Duroquinol at a final concentration of 0.5 mM, and the \u0026lsquo;uncoupler\u0026rsquo; gramicidin (5 \u0026mu;M) were added to the reaction mixture to facilitate regeneration of reduced plastoquinone and cytochrome \u003cem\u003ef\u003c/em\u003e. The cell suspension was diluted to an optical density of 1.0 at 730 nm for spectrophotometric analysis, the output signal referenced to the pre-flash output function, and a noise suppression algorithm utilized to minimize noise in the signal and detector output.\u003c/p\u003e"},{"header":"Results ","content":"\u003cp\u003eThe pattern of absorbance and resulting spectral changes associated with light- and chemically - induced changes in the oxidation state of cytochrome \u003cem\u003ef\u0026nbsp;\u003c/em\u003ehas been documented in previous studies on spinach thylakoids and \u003cem\u003eC. reinhardtii\u003c/em\u003e (\u003cstrong\u003eCramer and Hasan, 2016\u003c/strong\u003e). \u0026nbsp;The present study extended this analysis to the redox changes of the cytochrome \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e\u003cem\u003ef\u0026nbsp;\u003c/em\u003ecomplex in\u0026nbsp;the cyanobacterium,\u0026nbsp;\u003cem\u003eSynechococcus\u003c/em\u003e \u003cem\u003esp.\u003c/em\u003e PCC 7002.\u0026nbsp;The light-induced\u0026nbsp;redox changes were referenced to those obtained by actinic Illumination of spinach chloroplasts, suspended at a concentration of 50-100\u0026nbsp;\u0026mu;g/ml chlorophyll with methyl viologen (0.5 mM), as the electron acceptor.\u0026nbsp;The peak of the spectrum generated to characterize the extent of the cytochrome-specific redox changes (\u003cstrong\u003eCramer and Hasan, 2016\u003c/strong\u003e; \u003cstrong\u003eKramer and Crofts, 1990\u003c/strong\u003e) is close to 554 nm (\u003cstrong\u003eFig. 1A\u003c/strong\u003e), the\u0026nbsp;\u0026alpha;- band (in green region of spectrum)-peak of the cytochrome \u003cem\u003ef\u003c/em\u003e redox difference spectrum.\u003c/p\u003e\n\u003cp\u003eA spectrum of the light-induced absorbance change, an example shown in \u003cstrong\u003eFig. 1B\u003c/strong\u003e, was generated from the\u0026nbsp;set of individual absorbance\u0026nbsp;measurements at (\u003cstrong\u003eFig. 1A\u003c/strong\u003e). The peak of the difference spectrum as defined by \u0026lsquo;best fit\u0026rsquo; analysis shows that the spectrum peaks at 555 nm. The difference spectrum for oxidation of heme \u003cem\u003eb\u003c/em\u003e in the \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e\u003cem\u003ef\u003c/em\u003e complex was centered at 563-564 nm (\u003cstrong\u003eFig. 1B)\u003c/strong\u003e. These spectra are consistent with the well-documented precedent for the spectrum for light-induced oxidation of cytochrome \u003cem\u003ef\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eCramer and Hasan, 2016; Hasan \u003cem\u003eet al\u003c/em\u003e., 2018\u003c/strong\u003e),\u003cem\u003e\u0026nbsp;\u003c/em\u003eand also provide a spectrophotometric reference for the additional studies reported here on the amplitude and kinetics of the light-induced redox turnover of \u003cem\u003eb\u003c/em\u003e-cytochromes in intact cyanobacteria. The amplitude and time course of \u0026lsquo;dark reduction,\u0026rsquo; the redox change in the dark \u003cu\u003eto\u0026nbsp;\u003c/u\u003elight-induced oxidation of cytochrome \u003cem\u003ef\u003c/em\u003e in spinach thylakoid membranes, measured by the absorbance change at 554 nm relative to a reference change measured at 540 nm, is shown (\u003cstrong\u003eFig. 2A).\u003c/strong\u003e The first 40 ms in the display show a baseline which displays (\u003cstrong\u003ei\u003c/strong\u003e) the background noise in the measurement followed by (\u003cstrong\u003eii\u003c/strong\u003e) a spike arising from leak into the detector of the light flash at approximately 40 ms into the recording In the absence or presence of a sample. The amplitude of the effective absorbance change associated with a leak of the relatively intense actinic light flash, if displayed alone, would decrease immediately after termination of the light flash. However, a relatively slow decrease in absorbance is seen with a half-life of 8-10 msec which arises from the absorbance change associated with \u003cstrong\u003e(i)\u0026nbsp;\u003c/strong\u003ethe light-induced oxidation, the detailed time course not resolved here, and \u003cstrong\u003e(ii)\u0026nbsp;\u003c/strong\u003ethe subsequent \u0026lsquo;dark reduction\u0026rsquo; of cytochrome \u003cem\u003ef\u003c/em\u003e after termination of the light flash. The time course of the initial light-induced oxidation of the cytochrome was not resolved in these measurements.\u003c/p\u003e\n\u003cp\u003eUnder the same experimental conditions, similar measurements were done at a measuring light wavelength of 563 nm to determine the spectral properties of the heme \u003cem\u003eb\u003csub\u003e6\u003c/sub\u003e\u003c/em\u003e redox change (\u003cstrong\u003eFig. 2B\u003c/strong\u003e). The reaction medium contained duroquinol as a reductant of the quinone pool and gramicidin as an uncoupler of electron transfer. \u0026nbsp;A \u0026lsquo;slow\u0026rsquo; oxidation of heme \u003cem\u003eb\u003c/em\u003e, consistent with prediction and previous observations was observed. Presumably because of the difference in rateconstants for oxidation and reduction, the change in absorbance for heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eis significantly smaller than that of cytochrome \u003cem\u003ef\u003c/em\u003e. The critical experiment which describes the presence of these absorbance changes in intact cells, spectrophotometric measurement at the alpha-band absorbance peaks associated with cytochromes \u003cem\u003ef\u0026nbsp;\u003c/em\u003eand \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e, is shown (\u003cstrong\u003eFigs. 3A, B)\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003e1. Growth of Cyanobacterial\u003c/b\u003e \u003cb\u003eCultures\u003c/b\u003e. \u003cem\u003eSynechococcus sp.\u003c/em\u003e PCC 7002, wild-type, were grown in medium A (\u003cb\u003eHasan\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e., \u003cb\u003e2018\u003c/b\u003e) under optimal growth conditions, 38\u0026deg;C with an incident light intensity of 100\u0026ndash;150 micro Einsteins\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 2% (v/v) CO\u003csub\u003e2\u003c/sub\u003e/air. The cell titer was assayed using a Cary 3 spectrophotometer through the optical density of the cell culture measured at 660 nm relative to that at a reference wavelength of 730 nm, using a spectrophotometer with the light collection and detector geometry designed for a high efficiency of collection of light scattered from the turbid cell suspension.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2\u003c/b\u003e, \u003cb\u003ePreparation of Spinach Chloroplast Thylakoid Membranes\u003c/b\u003e. Thylakoid membranes were prepared according to (\u003cb\u003eHasan and Cramer (2014); Hasan et al., 2014).\u003c/b\u003e Spinach leaves (15 g) were homogenized (in the presence of 100 ml of buffer A or grinding buffer at high speed for 30 seconds. The resulting mixture was passed through a cloth filter. The filtrate was then separated by centrifugation (2 min, 2,000 x g, 4\u003csup\u003eo\u003c/sup\u003eC), and the isolated pellet resuspended in \u0026lsquo;buffer B\u0026rsquo; (\u003cb\u003eCramer and Hasan, 2016\u003c/b\u003e) and incubated on ice for 10 min. The solution was centrifuged twice at 4000 rpm for 10 min, and the resulting sediment stored in buffer B containing protease inhibitors. Aliquots of this suspension were used to carry out determinations of chlorophyllconcentration.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3\u003c/b\u003e. \u003cb\u003eFlash Kinetic Spectroscopy\u003c/b\u003e. To measure cytochrome \u003cem\u003ef\u003c/em\u003e oxidation, wild type \u003cem\u003eSynechococcus\u003c/em\u003e was used at a final chlorophyll concentration of 50\u0026ndash;100 \u0026micro;g/ml in the presence of 0.5 mM methyl viologen (MV) as the terminal electron acceptor. Flash-induced oxidation of cytochrome \u003cem\u003ef\u003c/em\u003e, using a triggered Xenon flash single-beam spectrometer (\u003cb\u003eZhang\u003c/b\u003e \u003cb\u003eet al.\u003c/b\u003e, \u003cb\u003e2008\u003c/b\u003e) was measured at 554 nm relative to 540 nm as a reference wavelength, thus generating the difference absorbance signal, ΔA\u003csub\u003e554\u0026ndash;540\u003c/sub\u003e. An \u0026lsquo;action spectrum\u0026rsquo; which describes the light-induced oxidation of cytochrome \u003cem\u003ef\u003c/em\u003e was obtained by repeating this measurement over the wavelength range (548\u0026ndash;560 nm) that spans the cytochrome alpha-band spectral domain. The same procedure was used to obtain an action spectrum associated with the light-induced redox change for heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e reduction in spinach thyla-\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is shown here that a major difference exists between the results of visible light flash excitation experiments on the \u003cem\u003eb\u003c/em\u003e-hemes obtained (\u003cstrong\u003ea\u003c/strong\u003e) with thylakoid (e. g., spinach) membranes and (\u003cstrong\u003eb\u003c/strong\u003e) cyanobacteria, represented here by the studies on \u003cem\u003eSynechococcus\u003c/em\u003e (\u003cstrong\u003eFig. 2B\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;3B,\u003c/strong\u003e respectively\u003cstrong\u003e)\u003c/strong\u003e. Thus, based on a measured absorbance change of approximately 1.5 x 10\u003csup\u003e-3\u003c/sup\u003e for cytochrome f at 554 nm (\u003cstrong\u003eFig. 3A\u003c/strong\u003e), a significant amplitude for photoreduction of heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e, assayed through the magnitude of the absorbance difference at 563-564 nm, was not detected. The detector of the spectrophotometric device has an intrinsic noise level of 0.5 x 10\u003csup\u003e-4\u003c/sup\u003e absorbance units. Thus, if there is a light-induced change in the \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u0026nbsp;\u003c/sub\u003eheme, it is substantially smaller than would be expected for any significant amplitude of reduction.\u003c/p\u003e\n\u003cp\u003eIn summary, the ratio of the light-induced absorbance change between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u0026nbsp;\u003c/sub\u003eand cytochrome \u003cem\u003ef\u003c/em\u003e is significantly smaller than 0.5, partly a consequence of the structural proximity of\u0026nbsp;cytochrome \u003cem\u003ef\u003c/em\u003e to the photosystem I reaction center.\u0026nbsp;These studies suggest an alternate pathway and mechanism for energy transduction linked to electron transport in the \u0026lsquo;main\u0026rsquo; electron transport chain linking the two photosystems. Regarding the location of this additional site for energy transduction, It has been proposed that a significant component of the proton electrochemical potential gradient in oxygenic photosynthesis could be provided through a pathway and mechanism of proton translocation involving conformation changes of the Rieske protein present in the mitochondrial cytochrome \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex (\u003cstrong\u003ePapa \u003cem\u003eet al\u003c/em\u003e.,\u003c/strong\u003e \u003cstrong\u003e1983).\u003c/strong\u003e The latter model combines \u0026lsquo;proton-motive\u0026rsquo; catalysis by quinone bound to the cytochrome \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex, and proton conduction along intra-membrane, trans-membrane pathways in the apoproteins of the cytochrome \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAcknowledgments\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWACproposedthe study; AP carried out the spectrophotometric analysis as part of an undergraduate research project. The authorsthank the College of Science. Purdue University, for providing a summer research Fellowship to AP, and the Department of Biological Sciences, for providing technical support of the facilities, with no competing interests. \u0026nbsp;We thank Ms. Rebecca Harding for assistance in the assembly and submission of the manuscript. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eMitchell, P. (1976) Possible Molecular Mechanisms of the Proton Motive Function of Cytochrome Systems. J. Theor. Biol, 62, 327\u0026ndash;367.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eE. C. Slater (1983) The \u0026lsquo;Q cycle,\u0026rsquo; A Ubiquitous Mechanism of Electron Transfer. Trends Biochem. Sci., 8, 239\u0026ndash;242.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSchneider, H., J. J. Lemasters, and C. R. Hackenbrock (1985) Membrane fluidity and mobility of ubiquinone. In \u003cem\u003eCoenzyme Q\u003c/em\u003e (ed. (G. Lenaz, ed.), pp.\u0026nbsp;201\u0026ndash;204. John Wiley, New York.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTrumpower, B. L. (2002) A concerted alternating sites mechanism of ubiquinol oxidation by the cytochrome \u003cem\u003ebc\u003c/em\u003e (1) complex. Biochim Biophys Acta, 1555, 166\u0026ndash;173.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCrofts, A. R., S. W. Meinhardt (1983) The role of the quinone pool A modified Q-cycle mechanism for the cyclic electron-transfer chain of \u003cem\u003eRhodopseudomonas sphaeroides\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCrofts, A. R. S. W. Meinhardt, and K. B. Jones (1983) The role of the quinone pool in the cyclic electron transfer chain of \u003cem\u003eRhodopseudomonas sphaeroides\u003c/em\u003e: a modified Q cycle mechanism. Biochim Biophys Acta, 723, 202\u0026ndash;218.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJoliot, P., and A. Joliot (2002) Cyclic electron transfer in plant leaf. \u003cem\u003eProc. Natl. Acad Sci., U.S\u003c/em\u003e, 99, 10209\u0026ndash;10214.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCramer, W. A. and D. B. Knaff (1990) \u0026lsquo;\u003cem\u003eThe Quinone Connection\u003c/em\u003e, chapt. 5 in \u003cem\u003eEnergy Transduction in Biological Membranes\u003c/em\u003e, pp.\u0026nbsp;545, a textbook, Springer-Verlag, ISBN 0-387-96761-3.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCramer, W. A. and W. L. Butler (1967) Light induced absorption changes of two cytochrome \u003cem\u003eb\u003c/em\u003e components in the electron transport chain of spinach chloroplasts. Biochim. Biophys. Acta 143, 332\u0026ndash;339.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCramer, W. A. and S. Saif Hasan (2016) \u003cem\u003eThe Cytochrome b\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e\u003cem\u003ef Lipoprotein Complex\u003c/em\u003e; pp.\u0026nbsp;177\u0026ndash;207 in \u0026ldquo;\u003cem\u003eCytochrome Complexes: Evolution, Structures, Energy Transduction, and Signaling\u003c/em\u003e,\u0026rdquo; Eds. W. A. Cramer and T. Kallas, volume\u0026nbsp;41 in the Series \u0026quot;\u003cem\u003eAdvances in Photosynthesis and Respiration\u003c/em\u003e,\u0026rdquo; eds. Govindjee and T. Sharkey\u0026quot; (Springer, Dordrecht).ISSN 1572 \u0026ndash; 0233; ISBN 978-94-017-7479-6; DOI \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-94-017-7481-9\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKramer, D. M., and A. R. Crofts (1990) A modified Q cycle mechanism for electron transfer in chloroplasts. \u003cem\u003eCurrent Research in Photosynthesis\u003c/em\u003e, \u003cem\u003eProc. VIII Int. Conf. Photosynthesis\u003c/em\u003e, 2189\u0026ndash;2192.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePapa, S., M. Lorusso, D. Boffoli, and E. Bellowmo (1983) Redox-linked proton translocation in the \u003cem\u003eb\u0026ndash;c\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex from beef-heart mitochondria reconstituted into phospholipid vesicles: general characteristics and control of electron transport. European Journal of Biochemistry 137, 405\u0026ndash;412.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHasan, S. S., D. Baniulis, E. Yamashita, M. V. Zhalnina, S. D. Zakharov, J. T. Stofleth, and W. A. 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Cramer (2014) A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex. J Phys Chem B 118, 6614\u0026ndash;6625.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhang, S., S. Hasan, and W. A. Cramer (2008) Structure - Function of the Cytochrome b6f Complex. Photochemistry and Photobiology, 84, 1349\u0026ndash;1358\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCramer, W. A. (2019) Structure-Function of the Cytochrome \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e\u003cem\u003ef\u003c/em\u003e Lipoprotein Complex: A Scientific Odyssey and Personal Perspective. Photosyn Res. 139, 52\u0026ndash;65\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHasan S. S., S. D. Zakharov, A. Chauvet, V. Stadnytskyi, S. Savikhin and W. A. Cramer (2014) A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex. J Phys Chem B 118, 6614\u0026ndash;6625\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePapa, S., M. Lorusso, D. Boffoli, and E. Bellowmo (1983) Redox-linked proton translocation in the \u003cem\u003eb\u0026ndash;c\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex from beef-heart mitochondria reconstituted into phospholipid vesicles. Eur. J. Biochem. 137: 405\u0026ndash;412.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBaniulis, D., E Yamashita, H Zhang, S Saif Hasan, WA Cramer (2014). Photochemistry and conservation of lipid functions in cytochrome \u003cem\u003ebc\u003c/em\u003e complexes. Structure 22, 1008\u0026ndash;1015,\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3463667/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3463667/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA working concept for the regulation of electron transport in oxygenic photosynthesis is that the electron transfer rate between the two photosystems, PSI and PSII, is governed by a ‘Q-cycle’ pathway operating in the electron transport chain which connects the two ‘reaction center’ complexes. The ‘Q-cycle’ concept was initially inferred from studies on mitochondrial electron transport. This concept has been assumed to be relevant to the electron transport pathways operating in oxygenic photosynthesis, with the majority of studies done on chloroplasts or thylakoid membranes The present study examines the existence and properties of a putative ‘Q-cycle’ in cyanobacteria. Light-induced spectral changes associated with cytochrome redox reactions in intact cells of the cyanobacterium \u003cem\u003eSynechococcus sp\u003c/em\u003e. corresponded to the oxidation-reduction of cytochrome \u003cem\u003ef\u003c/em\u003e. \u0026nbsp;A correlated reduction of heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e was, however, not observed. The absence of significant cytochrome \u003cem\u003eb\u003c/em\u003e reduction might be considered inconsistent with the set of electron transfer events associated conceptually with a ‘Q-cycle’ model of the electron transfer events in the chain. However, because heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e in the intact cyanobacteria is mostly reduced, it is not observable as a net electron acceptor of the plastoquinol or semiquinone formed by electron transfer from photosystem II. The redox environment of intact cyanobacteria in the dark resting state has an ambient potential sufficiently reducing that the ‘Q-cycle’ pathway for electron transport, well studied and characterized for function in isolated thylakoid membranes or chloroplasts, is not observed. This apparent quandary’ is a consequence of the reducing (negative potential) intracellular redox environment of cyanobacteria, which imposes a reduced state on the \u003cem\u003eb\u003c/em\u003e-hemes, thereby preventing observation of their light-induced reduction.\u003c/p\u003e","manuscriptTitle":"Concerning the Application of the Q Cycle to Electron Transport in Cyanobacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-24 17:07:52","doi":"10.21203/rs.3.rs-3463667/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e521b82-19af-4ece-b6ec-a83310a763e7","owner":[],"postedDate":"October 24th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-06T08:53:55+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-24 17:07:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3463667","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3463667","identity":"rs-3463667","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}