Pedro J. Aparicio

Flavin type action spectrum of nitrate utilization by Monoraphidium braunii

The light‐dependent utilization of nitrate by the green alga Monoraphidium braunii, coming from nocturnal dark periods, shows an action spectrum of flavin type with two main bands: one in the blue, peaking at 450 and 480 nm, and the other in the near‐UV region with a maximum at 365 nm. Other results indicate that cells growing on nitrate as the only nitrogen source resynthesize nitrate reductase daily, which implies the nocturnal loss of this enzyme. The biosynthesis of nitrate reductase at the beginning of the light periods can proceed under red light. In addition, blue or near‐UV light is required for the activation of the previously formed nitrate reductase.

Blue Light Requirement for HC03 Uptake and Its Action Spectrum in Monoraphidium braunii

The uptake and assimilation of HCO3 by the green unicellular alga Monoraphidium braunii can be monitored by the alkalinization of the external medium or by the O2 evolution associated with the uptake and reduction of this anion. The activation of HCO3 uptake in this microalga required the irradiation of the cell suspensions with low photon fluence rates of short wavelength radiation. Thus, when the cells were irradiated with strong red light in the presence of HCO3, very little alkalinization of the external medium or O2 evolution could be observed. The O2 evolution rates measured under red light could be due to the assimilation of the CO2 derived from the HCO3 present in the medium. The blue light‐dependent O2 evolution rates were not diminished by a periplasmic carbonic anhydrase inhibitor, suggesting that HCO3 ‐dependent O2 evolution was due to the photoactivation of a selective HCO3 uptake system at the plasma membrane. The action spectrum for HCO3‐ uptake in M. braunii was very similar to those reported for NO3‐ and CI‐ suggested that a flavoprotein may be the photoreceptor for this response.

Photoinactivation of the detoxifying enzyme nitrophenol reductase from Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus E1F1 detoxifies 2,4-dinitrophenol by inducing an NAD(P)H-dependent iron flavoprotein that reduces this compound to the less toxic end product 2-amino-4-nitrophenol. This nitrophenol reductase was stable in crude extracts containing carotenes, but it became rapidly inactivated when purified protein was exposed to intense white light or moderate blue light intensities, especially in the presence of exogenous flavins. Red light irradiation had no effect on nitrophenol reductase activity. Photoinactivation of the enzyme was irreversible and increased under anoxic conditions. This photoinactivation was prevented by reductants such as NAD(P)H and EDTA and by the excited flavin quencher iodide. Addition of superoxide dismutase, catalase, tryptophan or histidine did not affect photoinactivation of nitrophenol reductase, thus excluding these reactive dioxygen species as the inactivating agent. Substantial protection by 2,4-dinitrophenol also took place when the enzyme was irradiated at a wavelength coinciding with one of the absorption peaks of this compound (365nm). These results suggest that the lability of nitrophenol reductase was due to the absorption of blue light by the flavin prosthetic group, thus producing an excited flavin that might irreversibly oxidize some functional group(s) necessary for enzyme catalysis. Nitrophenol reductase may be preserved in vivo from blue light photoinactivation by the high content of carotenes and excess of reducing equivalents in phototrophic growing cells.

Effects of light quality, CO2 tensions and NO 3 +− concentrations on the inorganic nitrogen metabolism of Chlamydomonas reinhardii

The blue light dependent utilization of nitrate by green algae under common air and high irradiances, besides its assimilatory nature, is associated with the release of NO2− and NH4+ to the culture medium. If the CO2 content of the sparging air was increased up to 2%, previously excreted NO2− and NH4+ were rapidly assimilated. When under air and high irradiances the cell density in the culture reached values corresponding to 25 μg Ch 1.ml-1, no further growth was observed and the highest values of NO3− consumption and NO2− and NH4+ release were attained. Besides low CO2 tensions, increasing NO3− concentrations in the medium stimulated the release of NO3− and NH4+. Under CO2-free air the consumption of NO3− and the release of NO2− and NH4+ on a total N bases were almost stoichiometric and their rates saturated at much lower irradiances than under air. Under CO2-free air high rates of NO2− release were only observed under the blue radiations that were effectively absorbed by photosynthetically active pigments, i.e. 460 nm, but not under 404 and 630 nm radiations. However, the simultaneous illumination of the cells with 404 and 630 nm monochromatic light showed a remarkable synergistic effect on NO2− release.The results are discussed in terms of the close relationship between C and N metabolism, the photosynthetic reducing power required to convert NOinf3sup±-N into R − NH2-N and the blue light activation of nitrate reductase.

EFFECTS OF SHORT PULSES OF BLUE LIGHT ON THE ALKALINIZATION ASSOCIATED WITH THE UPTAKE OF NO3− AND CL− BY THE GREEN ALGA MONORAPHIDIUM BRAUNII AND RELATED ACTION SPECTRA

In Monoraphidium braunii, uptake of NO3−, NO2− and Cl− is associated with proton transport and triggered by blue light (BL). Only 10 s after cells able to reduce NO3− to NH4+ were irradiated with continuous, low‐fluence BL in the presence of NO3−, an alkalinization of the medium began and only became interrupted by switching off the BL with a 60–90 s time lag. With 30 s BL pulses, the NO3−‐dependent alkalinization lasted 3–5 min until it stopped. When the cells were exposed to continuous BL in the presence of Cl−, the alkalinization also started within 10 s but lasted only 3 min. After that, the pH remained constant and decreased when the BL was switched off. With 30 s BL pulses, the Cl−‐dependent alkalinization lasted 3 min and then decreased to its initial value. The NO3−‐dependent alkalinization shown by cells unable to reduce NO3− to NH4+ was similar to that observed in the presence of Cl−. These alkalinization rates fit the Bunsen‐Roscoe reciprocity law. With 2 s pulses of high‐fluence BL, the delay time of the NO3 ‐ or Cl−‐dependent alkalinizations was only 2 s, one of the fastest BL responses reported so far. The action spectra for Cl− and NO3− uptakes proved to be very similar and matched the absorption spectra of flavins, including the 267 nm peak.