Distinguishing the Roles of Thylakoid Respiratory Terminal Oxidases in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

Various oxygen-utilizing electron sinks, including the soluble flavodiiron proteins (Flv1/3), and the membrane-localized respiratory terminal oxidases (RTOs), cytochrome c oxidase (Cox) and cytochrome bd quinol oxidase (Cyd), are present in the photosynthetic electron transfer chain of Synechocystis sp. PCC 6803. However, the role of individual RTOs and their relative importance compared with other electron sinks are poorly understood, particularly under light. Via membrane inlet mass spectrometry gas exchange, chlorophyll a fluorescence, P700 analysis, and inhibitor treatment of the wild type and various mutants deficient in RTOs, Flv1/3, and photosystem I, we investigated the contribution of these complexes to the alleviation of excess electrons in the photosynthetic chain. To our knowledge, for the first time, we demonstrated the activity of Cyd in oxygen uptake under light, although it was detected only upon inhibition of electron transfer at the cytochrome b6f site and in ∆flv1/3 under fluctuating light conditions, where linear electron transfer was drastically inhibited due to impaired photosystem I activity. Cox is mostly responsible for dark respiration and competes with P700 for electrons under high light. Only the ∆cox/cyd double mutant, but not single mutants, demonstrated a highly reduced plastoquinone pool in darkness and impaired gross oxygen evolution under light, indicating that thylakoid-based RTOs are able to compensate partially for each other. Thus, both electron sinks contribute to the alleviation of excess electrons under illumination: RTOs continue to function under light, operating on slower time ranges and on a limited scale, whereas Flv1/3 responds rapidly as a light-induced component and has greater capacity.

Figure 1.

Schematic diagram of the thylakoid membrane-localized photosynthetic and respiratory electron transfer chains. Lines indicate electron transport; dotted lines indicate possible but poorly characterized electron transfer and proton pathways. Cox, Cytochrome c oxidase; Cyd, cytochrome bd quinol oxidase; Cyt c₆, cytochrome c₆; Fd, ferredoxin; Flv2/4, flavodiiron proteins 2/4; Flv1/3, flavodiiron proteins 1/3; FNR, ferredoxin-NADP⁺ oxidoreductase; NDH-1, NAD(P)H dehydrogenase-like complex type 1; NDH-2, NAD(P)H dehydrogenase type 2; Pc, plastocyanin; PQ, plastoquinone; PQH₂, plastoquinol; SDH, succinate dehydrogenase.

Figure 2.

Rates of oxygen exchange in wild-type (WT) and ∆cyd mutant cells incubated in darkness for 5 min and then illuminated with a strong white light (400 μmol photons m⁻² s⁻¹) for the next 5 min. Black bars and gray bars indicate the rates of total oxygen uptake by cells in darkness and in the light, respectively; white bars indicate the gross oxygen production rate. Measurements were performed either in the absence of inhibitors (control) or in the presence of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) or DBMIB + 2-N-heptyl-4-hydroxyquinoline N-oxide (HQNO). Values are means ± sd; n = 3 to 5.

Figure 3.

Rates of total oxygen uptake in darkness (black bars) and in the light (gray bars) in the wild type (WT) and ∆flv1/3 acclimated for 3 d to a fluctuating light FL 20/500 regime (20 μmol photons m⁻² s⁻¹ background light interrupted by 30-s pulses of 500 μmol photons m⁻² s⁻¹ light every 5 min). Measurements were performed using MIMS on cells incubated in darkness for 5 min and then illuminated with a strong white light (400 μmol photons m⁻² s⁻¹) for 5 min either in the absence (control) or in the presence of HQNO. Values are means ± sd, n = 3. The asterisk indicates a statistically significant difference between measurement with HQNO compared with control samples (P < 0.05).

Figure 4.

Growth of the Synechocystis wild type (WT) and RTO-deficient mutants under FL 20/500 (20 μmol photons m⁻² s⁻¹ background light interrupted every 5 min with 30-s high light pulses of 500 μmol photons m⁻² s⁻¹; A) or 5-min-dark/5-min-high light (200 μmol photons m⁻² s⁻¹; B) square-wave cycles monitored by optical density (OD) measurements at 750 nm. Values are means ± sd, n = 3.

Figure 5.

MIMS analysis of oxygen uptake by PSI-less mutant cells in the absence (control) and presence of HQNO and HQNO plus KCN. Oxygen uptake was monitored for 5 min in darkness and 5 min under a light intensity of 150 μmol photons m⁻² s⁻¹. Arrows indicate the beginning of illumination. The slope of the curves does not provide a precise quantitative measure of the rate of oxygen consumption until it is corrected for the isotopic ratio (¹⁶O/¹⁸O).

Figure 6.

Rapid light curves of the wild type (WT) and RTO-deficient mutants: effective yield of PSII [Y(II); A] and donor side limitation of PSI [Y(ND); B]. Values are means ± sd, n = 3. Asterisks indicate statistically significant differences compared with the wild type (P < 0.05).

Figure 7.

Fluorescence analysis of wild-type (WT) and RTO-deficient mutant cells. Fluorescence was recorded in darkness (F₀; black bars on the time scale) and under far-red (FR) light. Saturating pulses indicated by flashes were fired to monitor F_m^D and maximum fluorescence level under the FR background (F_M^FR). The values are provided in Supplemental Table S1. Samples were adjusted to a Chl concentration of 15 μg mL⁻¹ and dark adapted for 10 min before the measurements. A representative curve of three independent experiments is shown. r.u., Relative units.

Figure 8.

Relaxation of the flash-induced fluorescence yield in darkness. Q_A⁻ reoxidation was monitored from dark-adapted wild-type (WT; black squares), ∆cox (red circles), ∆cox/cyd (green diamonds), and ∆cox/cyd (purple diamonds) cells preilluminated with FR light for 30 s (A) and in the presence of 20 μm DCMU (B). Values are means ± sd, n = 3. The F₀ and F_m values were normalized to 0 and 1, respectively, to facilitate comparison of the kinetics. r.u., Relative units.

Figure 9.

P700 oxidoreduction. P700 oxidation and rereduction in wild-type and mutant cells was illuminated with strong FR light for 5 s. Cells are as follows: wild type (black), ∆cox (blue), ∆cyd (green), and ∆cox/cyd (red). Curves were normalized to the same amplitude to facilitate comparison of the kinetics. Representative curves of three independent experiments are shown. r.u., Relative units.