Kinetics and Apparent K m of Oxygen Cycle under Conditions of Limiting Carbon Dioxide Fixation

Abstract

A mass spectrometer with a membrane inlet was used to monitor light-driven 02 evolution, 02 uptake, and CO2 uptake in suspensions of algae (Scenedesmus obHlquus). We observed the following. (a) The rate of 02 uptake, which, in the presence of iodoacetamide, replaces the uptake of CO2, showed a distinct plateau (Vm..) beyond-30% 02 and was half-maximal at-8% 02. We concluded that this light-driven 02 uptake process, which does not involve carbon compounds, is saturated at lower 02 concentrations than are photorespiration and glycolate formation. (b) In the absence of inhibitor, 02 evolution was relatively unaffected by the presence or absence of C02. During the course of CO2 depletion, electron flow to C02 was replaced by an equivalent flow to 02. (c) There was a distinct delay between the cessation of C02 uptake and the increase in 02 uptake. We ascribe this delay to the transient utilization of another electron acceptor-possibly bicarbonate or another bound form of CO2. Light-induced 02 uptake in green plants appears to be due to several processes (e.g. ref. 3). In a previous communication (7) we reported that illuminated algae can reduce 02 at a high rate under conditions in which CO2 fixation is not occurring. For example, during the lag in CO2 reduction following a dark-light transition, 02 evolution occurs at a high rate but is compensated by a concomitant 02 uptake. A high rate of 02 uptake, fully compensating 02 evolution, is also observed in the presence of inhibitors of CO2 fixation, which indicates that a special high capacity oxidase distinct from ribulose-diP oxygenase exists in whole cells. In this communication, we describe experiments in which we determined the 02 affinity of the oxidase involved in this 02 cycle, and the kinetics of 02 uptake under low CO2 conditions. A preliminary report of this work was presented earlier (6). MATERIALS AND METHODS The mass spectrometer inlet system, data acquisition system, and assay procedures used in these experiments were similar to those described earlier (7, 8). Algae were suspended in 0.1 M phosphate buffer (pH 6). The pertinent cell densities are given in the figure legends. The values for 02 uptake (Uo) and evolution (Eo) were obtained from the expressions: Uo = A1802 (I + 1602/1802) Eo = A'602IA'802 (1602/1802) Values for CO2 uptake (Uc) were obtained from the sum of the uptakes of '3CO2 and '2CO2. The argon added during the course ' of the 02-CO2 depletion procedures served as an internal standard. Gaseous 1802 (99 atom %) was obtained from Bio-Rad Laboratories. '3C-Labeled barium carbonate (95 atom %) was obtained from Merck, Sharp & Dohme. Cultures of Scenedesmus obliquus (Gaffron strain D3) were grown under the conditions described previously in this laboratory (2). Ten mm NH4' was used as the nitrogen source to avoid interference due to nitrate reduction. Due to an unavoidable mass spectrometer background at m/e = 44 ('2CO2), measurements of low CO2 concentrations necessitated the use of 13C-labeled algae. To this end, Scenedesmus cells were grown in a closed culture vessel containing 13CO2 generated from 13C-labeled barium car-bonate (the initial concentration of CO2 in the gas phase was 5%). Chl concentrations were determined by the methods described in (2). RESULTS AND DISCUSSION Substrate Affinity of 03 Cycle (Apparent Kin). As described earlier (7), in the presence of the Calvin cycle inhibitor, iodoacet-amide, 02 uptake replaces CO2 uptake and balances 02 evolution, so that there is no net change in 02 concentration. This inhibitor offers the opportunity to study the 02 uptake reaction unemcum-bered by the varying and complicating effects of CO2. Figure 1, which is a compilation of a series of experiments similar to those of Figure 2 in reference 7, shows the net rates of 02 uptake (and 02 evolution, the two are mirror images) by Scenedesmus D3 in the presence of iodoacetamide. Note that at 02 concentrations above 30% the rate is maximal and invariant within experimental error (in Fig. 1, Vm. = 33.5 cell vol/hr). Half this rate is observed at an atmospheric abundance of 8% (indicated as Ki, Fig. 1). Five repetitions ofthis set ofexperiments (all made at room temperature and at intensities inducing greater than half-maximal rates) gave similar results. The linear plot (v versus v/02) in the inset of Figure I and the dashed curve with its computed maximum rate value (V,,ax = 40 cell vol/hr) and half-value (K,m = 11%) show an attempt to fit the data with a rectangular hyperbola. Such a plot is customarily associated with Michaelis-Menten kinetics, the mechanistic basis for the notation K,. This function describes the data rather poorly, particularly the sudden transition to a well defined saturation plateau. A much better fit is obtained by an exponential function (solid line in Fig. 1). It might be interesting to note that similar saturation curves have been reported for CO2 and light intensity (5). Unfortunately, however, with properly adjusted parameters, other (hyperbolic) functions can describe the data equally well and within experimental error. The half-saturation value of 8% obtained in these experiments is significantly different from the half-saturation value(s) reported for photorespiration and glycolate formation. According to reference 10 (see also 4), the latter processes are not rate-saturated even at 100%o (I atm) of 02-Invariance of Electron Flow during Course of CO2 Depletion. Figure 2 shows the kinetics of the gas exchange reactions during the final phase of CO2 depletion (the light was turned on 4 min 915