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Flavodiiron-Mediated O2 Photoreduction Links H2 Production

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Flavodiiron-Mediated O2 Photoreduction Links H2 Production A PAM Vacuum line to the spectrometer Thermostated Cuvette Magnetic stirrer PAM measurements B (PSII activity) Optic fiber Water jacket Light supply Cuvette Vacuum line to the spectrometer Teflon membrane (Gas exchange) Supplemental Figure S1. Experimental device used for combined measurements of gas exchange by using MIMS and chlorophyll fluorescence with a PAM. A. Picture of the experimental device. B. Schematic view of the cuvette used for simultaneous measurements of fluorescence and mass spectrometry. 2 .0 A W T ) . u . 1 .5 r ( e c n e c s e F M ' r F M o 1 .0 u l f l l y F h p o r o l F s h 0 .5 C 0 .0 0 2 0 0 4 0 0 6 0 0 T im e (s ) 2 .0 B flv B -2 0 8 ) . u . 1 .5 r ( e c n e c s e F M ' r F M o 1 .0 u l f l l y F h p o r o l F s h 0 .5 C 0 .0 0 2 0 0 4 0 0 6 0 0 T im e (s ) Supplemental Figure S2. Representative chlorophyll fluorescence recordings obtained during a dark to light transient in anaerobically acclimated C. reinhardtii cells. (A) wild- type strain (WT). (B) flvB-208 mutant. Cells were harvested from photobioreactor cultures and placed in the MIMS cuvette. Chlorophyll fluorescence was recorded during a dark to light transient following 1h30 of dark anaerobic acclimation. Red spikes at the bottom of the figure show saturating flashes. 0 .8 0 .6 M F / 0 .4 F 0 .2 0 .0 0 .0 0 0 0 .0 0 5 0 .0 1 0 0 .0 1 5 -1 -1 Q u a n tu m y ie ld (m o l O 2 m ol p h o to n m g c h l) Supplemental Figure S3. Relationship between quantum yield of O2 evolution and ΔF/FM in aerobiosis at a low chlorophyll concentration. Chlorophyll fluorescence measurements were recorded using a PAM on top of a Hansatech cuvette linked to a Membrane Inlet Mass Spectrometer (MIMS). Recorded PSII yields were correlated with O2 18 exchange measurements made with O2 using the MIMS in aerobic conditions after 10 minutes dark adaptation. Experimental relation between the ΔF/FM and the quantum yield of O2 Evolution. In red is shown the linear regression for the points with a ΔF/FM lower than 0.6. Chlorophyll concentration was set to 10µg Chl.mL-1. 1 .0 ) 1 - n i n m o i l t h u l c 0 .5 o 1 v - e g 2 m O 2 t O e l N o 0 .0 m µ ( 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 -2 -1 L ig h t In te n s ity (µ m o l p h o to n m s ) Supplemental Figure S4. Saturation curve of net photosynthesis with green light. Net O2 evolution was measured with a MIMS after 1 min illumination at different green light intensities (mean +/- SD, n=5 biological replicates). The arrow indicates the light intensity used throughout this work. 2 .0 ) . u . r ( e 1 .5 * c * n * e * c * s e r o 1 .0 u l f W T l l y h flv B -2 0 8 p o 0 .5 r o flv B -3 0 8 l h C 0 .0 0 5 1 0 T im e (m in ) Supplemental Figure S5. Maximum fluorescence yield (FM’) upon a shift from dark anaerobiosis to light as a control for assessing PSII absorbance changes. Maximum fluorescence level during the first ten minutes of illumination. After 1h30 of dark anaerobic acclimation in the MIMS cuvette, algal suspensions of wild type (red line) and flvB mutant (grey and dark lines) strains were illuminated (660 µmol photons m-2 s-1 green light). Maximum chlorophyll fluorescence level was measured before illumination (FM) and each 30s upon illumination (FM’). Shown are mean values (+/-SD, n=3 for the mutants, n=6 for the WT), a star marks significant difference between the wt and the mutant strains (Pvalue <0.05) based on ANOVA analysis (Tukey adjusted P value). Fm’ value at the onset of light was normalized to 1 (r.u. relative units). A ___WT___ Loading (%) 100 50 100 100 100 100 100 100 kDa - 80 α-FlvA - 70 α-FlvB - 60 Ponceau B C 2 .5 ) 1 .0 . u ) . 1 2 .0 r - e ( t n i a e r c m n e 1 - e g 1 .5 l c n h s a C e h r g c o x 1 .0 m u e l 0 .5 l f o l D l h y d A 1 /A 2 / y m H h µ ( 0 .5 p h y d A 1 /A 2 flv B -1 o r o l h y d A 1 /A 2 flv B -2 h 0 .0 C h y d A 1 /A 2 flv B -3 T 2 -1 -2 -3 W /A B B B 1 lv lv lv 0 .0 A f f f d 2 2 2 0 5 0 1 0 0 1 5 0 y h /A /A /A 1 1 1 T im e (s ) A A A d d d y y y h h h Supplemental S6. Characterization of the mutant Chlamydomonas cells. (A) Immunodetection of Flvs in anaerobically acclimated cells. Chlamydomonas cells were harvested from exponential phase, centrifuged, resuspended at the same concentration in HSM medium and hermetically sealed in glass flack. Cells were harvested after 3 hours anaerobiosis and immunodetection was done on total cell extract. (B) Aerobic chlorophyll fluorescence pattern of triple and double mutant. Cells were grown in TAP medium under low light and harvested during exponential phase. Chlorophyll fluorescence measurements were performed using pulse amplitude modulated fluorimeter in the dark (black boxes) and under red actinic light (100 µmol -2 -1 photon m s ). Data are normalized on initial FM measurements. (C) In vivo hydrogenase activities as measured by H/D exchange rates after 1h dark anaerobic acclimation. Shown are mean values (+/-SD, n=2 for the mutants, n=4 for the WT). A 1 .5 ) . u . r ( e t a 1 .0 r e g n a h c 0 .5 x E D / H 0 .0 W T flv B -2 0 8 flv B -3 0 8 B ) 2 5 % ( n o 2 0 i t i b i h 1 5 n i e s a 1 0 n e g o 5 r d y H 0 W T flv B -3 0 8 Supplemental Figure S7. Hydrogenase activity assayed in vivo by H/D exchange. After 1h30 of dark anaerobic acclimation in the MIMS cuvette, algal suspensions of wild type (red box) and flvB mutant (grey and dark box) strains were saturated with Deuterium (D2). H2, HD and D2 were then recorded with a MIMS and H/D exchange rate was calculated (Jouanneau et al, 1980). (A) H/D exchange rates after dark anaerobic acclimation. Shown are mean values (+/-SD, n=2 for the mutants, n=4 for the WT). (B) Percentage of the inhibition of the H/D exchange rate after two minutes of illumination. Shown are mean values (+/-SD, n=2). 3 flv B -2 0 8 flv B -3 0 8 n o i 2 t a r ) t 2 n O e c M n µ o 1 ( c 2 O 0 -1 0 1 2 3 T im e (m in ) Supplemental Figure S8. Differences in O2 concentration present in the MIMS chamber containing flvB mutants compared to the WT during a dark to light transient in anaerobically acclimated C. reinhardtii. Differences in O2 concentration were calculated from absolute gas exchange as determined in Fig. 4. Shown are mean values of the following calculation for each of the two mutants : [O2](flvB)-[O2](WT) were [O2](flvB) and [O2](WT) are the O2 level in a mutant or a WT cell suspension respectively (+/-SD, n=3). 2 .0 ° W T A ab a flv B -3 0 8 ) . flv B -2 0 8 u . 1 .5 r ( b ° a e t a ° a a a" r a a a ' a "' a "' n a o i t 1 .0 u l c ' b "' o b" v b "' b "' b "' e b ' b" 2 O t e 0 .5 N 0 .0 in in in in in in in m m m m m m m 1 2 3 4 5 6 9 1 .5 B a * a * a * a ' a" a "' a "' a b ' b" 1 .0 b ' b "' b "' ) b" . b "' u b "' . r ( e 0 .5 t a r e k a 0 .0 t p u 2 O -0 .5 C t W T e N flv B -2 0 8 -1 .0 flv B -3 0 8 a a a -1 .5 in in in in in in m m m m m m 1 3 4 5 6 9 Supplemental Figure S9. Net O2 and CO2 exchange rates in the light in C. reinhardtii wild-type and two flvB mutants. After 95 min of dark anaerobic acclimation, WT (red) and two independent flvB mutant (flvB-208 and flvB-308, dark and grey respectively) cell -2 -1 suspensions were illuminated (660 µmol photons m s green light).
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