The photoreduction of H(2)O(2) by Synechococcus sp. PCC 7942 and UTEX 625

Abstract

It has been claimed that the sole H(2)O(2)-scavenging system in the cyanobacterium Synechococcus sp. PCC 7942 is a cytosolic catalase-peroxidase. We have measured in vivo activity of a light-dependent peroxidase in Synechococcus sp. PCC 7942 and UTEX 625. The addition of small amounts of H(2)O(2) (2.5 microM) to illuminated cells caused photochemical quenching (qP) of chlorophyll fluorescence that was relieved as the H(2)O(2) was consumed. The qP was maximal at about 50 microM H(2)O(2) with a Michaelis constant of about 7 microM. The H(2)O(2)-dependent qP strongly indicates that photoreduction can be involved in H(2)O(2) decomposition. Catalase-peroxidase activity was found to be almost completely inhibited by 10 microM NH(2)OH with no inhibition of the H(2)O(2)-dependent qP, which actually increased, presumably due to the light-dependent reaction now being the only route for H(2)O(2)-decomposition. When (18)O-labeled H(2)O(2) was presented to cells in the light there was an evolution of (16)O(2), indicative of H(2)(16)O oxidation by PS 2 and formation of photoreductant. In the dark (18)O(2) was evolved from added H(2)(18)O(2) as expected for decomposition by the catalase-peroxidase. This evolution was completely blocked by NH(2)OH, whereas the light-dependent evolution of (16)O(2) during H(2)(18)O(2) decomposition was unaffected.

Figure 1

H₂O₂-dependent quenching of Chl fluorescence (F) in Synecococcus sp. PCC 7942. The cells were dark-adapted for 10 min and the fluorescence signal was measured in the absence (O) and presence (F_o) of the weak, pulse-modulated measuring beam (MB). A single SF was given during this time, indicated by the transient increase in the F signal. The non-modulated WL was then turned on (120 μmol photon m⁻² s⁻¹, PAR). The cells were then allowed to deplete the medium of C_i, manifest as attainment of maximal fluorescence yield (F_M) during an SF, as described by Miller et al. (1991). The addition 50 μm C_i then resulted in reappearance of F quenching until this C _i was consumed. The additions of H₂O₂ then also resulted in F quenching, which was relieved as the H₂O₂ was decomposed, manifest as cessation of the H₂O-dependent O₂ evolution indicated by the traces below the F trace. SFs were periodically given so that qP could be estimated.

Figure 2

Quenching of Chl F due to addition of H₂O₂ solutions is not due to contaminant C_i. Synechococcus sp. PCC 7942 cells were allowed to deplete the medium of C_i described in Figure 1, then the response to an SF was monitored in the presence of: 500 μm C_i and about 280 μm O₂ (A); 500 μm C_i, 15 mm glycolaldehyde, and the Glc oxidase O₂ trap (B); or the latter adddition plus 50 μm H₂O₂ (C). F_v is defined as the difference between the F_M signal measured during an SF given while the cells were in WL after depleting the medium of C_i and the F_o signal measured with dark-adapted cells illuminated only by the weak MB (see Fig. 1).

Figure 3

Selective inhibition of in vivo catalase activity of 10 μm NH₂OH. A, 50 μm H₂O₂ was added to dark-adapted cells. O₂ evolution was monitored with or without subsequent addition of 10 μm NH₂OH. B, Cells were allowed to deplete the medium of C_i during illumination with WL (120 μmol photons m⁻² s⁻¹, PAR) and then F quenching and O₂ evolution were monitored after the addition of either 5 or 20 μm H₂O₂ in the absence or presence of 10 μm H₂O₂. For both A and B the maximum O₂ evolution rates, in terms of μmol O₂ mg⁻¹ Chl h⁻¹, are given beside the O₂ traces. F_v is as defined for Figure 2.

Figure 4

Decomposition of H₂O₂ in dark (A) and light (B) in response to H₂O₂ concentration. The maximum rate of O₂ evolution was used as the measure of H₂O₂ decomposition in the absence (□) or presence (⋄) of 10 μm NH₂OH. The rates for cells in the dark (A) were corrected for rates of O₂ uptake due to respiration measured just prior to H₂O₂ addition. For measurement of H₂O₂ decomposition in the light the cells were allowed to first deplete the medium of C_i. The difference between the rates of H₂O₂ decomposition in the light in the absence and presence of 10 μm NH₂OH is also given (○). The WL was 120 μmol photon m⁻² s⁻¹ (PAR).

Figure 5

Light-dependent H₂O₂ decomposition monitored as F quenching in Synechococcus sp. PCC 7942.Cells were allowed to deplete the medium of C_i in WL of 100 μmol photons m⁻² s⁻¹ (PAR) and then the degree of total F quenching was monitored after the addition of various concentrations of H₂O₂ in the absence (□) and presence (⋄) of 10 μm NH₂OH.

Figure 6

Decomposition of H₂¹⁸O₂ by Synechococcus sp. PCC 7942. Cells (12 μg Chl mL⁻¹) were incubated in the dark (A) or in WL (206 μmol photons m⁻² s⁻¹; B and C) in the absence (A and B) or presence of (C) of 50 μm NH₂OH. At the times indicated by the arrows, 50 μm H₂¹⁸O₂ was added and ¹⁶O₂ and ¹⁸O₂ evolution were monitored by MS. Cells incubated in the light were allowed to deplete the medium of C_i before addition of the H₂¹⁸O₂. A correction was made for ¹⁸O₂ contamination of the H₂¹⁸O₂ solution (see “Materials and Methods”).

Figure 7

Decomposition of H₂¹⁸O₂ by Synechococcus UTEX 625. Cells (11 μg Chl mL⁻¹) were incubated in the dark (A) or in WL (206 μmol photons m⁻² s⁻¹; B and C) in the absence (A and B) presence (C) of 50 μm NH₂OH. Conditions as described in Figure 6 except that 80 μm H₂¹⁸O₂ was added.