The Effects of Phenazine Dyes and N,N,N',N'-Tetramethyl-pphenylenediamine upon Light-induced Absorbance Changes and Photophosphorylation in Rhodospirillum rubrum Extracts

Abstract The effects of N-methylphenazonium methosulfate (PMS) have been compared with three other phenazine dyes and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). All accelerate the recovery of the initial absorbance changes in Rhodospirillum rubrum extracts induced by a 10-µsec flash, although only N-ethylphenazonium ethosulfate and TMPD stimulate photophosphorylation to the same extent as PMS. N-Ethyl-1,2-benzophenazonium perchlorate and N-ethyl-1,2,6,7-dibenzophenazonium chloride are less active. Absorbance changes with the dyes give evidence for the presence of at least three steps. The first is the light reaction, occurring at the instant of a flash (g25 µsec), generating an oxidant and a reductant. This is represented by the initial flash-induced absorbance change, which is unaffected by the presence of dyes. The second step, a slower reaction (complete in approximately 100 µsec with 0.5 mm reduced PMS), is the reduction of the photo-oxidant at the expense of reduced dye. This is evidenced by the marked stimulation of recovery of the initial flash-induced absorbance change by reduced dye, coupled with the appearance of oxidized dye (TMPD). The kinetics is monophasic first order, in contrast to the slow polyphasic kinetics of recovery in absence of dye. The initial rate of recovery of absorbance change is directly proportional to reduced dye concentration. The oxidation of TMPD induced by a single saturating 10-µsec flash is directly proportional to extract concentration and represents one-thirtieth of the bacteriochlorophyll content. Double flash experiments show that the initial flash-induced absorbance changes signal saturation of photochemical reaction centers. Maximal utilization of a second flash for dye photo-oxidation requires complete recovery of the initial absorbance changes induced by the first flash. The third step is the reduction of oxidized dye at the expense of reductant formed in the light reaction, completing the cyclic process of electron transport. Reduction of photo-oxidized TMPD is stimulated markedly by repetitive flashes. Reduction of TMPD following a single flash is very slow (t1/2 ∼ 1 sec), whereas reduction in repetitive flashes is complete in less than 50 msec (with a flashing rate of 20 per sec). The slowness of dye reduction compared to dye photo-oxidation is evidenced by the accumulation of oxidized dye during repetitive excitation. In continuous illumination the level of oxidized dye (∼20-fold that formed in by single 10-µsec flash) approximates the bacteriochlorophyll content. A study of photophosphorylation in flashing illumination indicates that addition of PMS or TMPD lowers the yield of ATP formed in a 10-µsec flash. With TMPD, the 10-µsec flash yield represents 0.05 ATP per electron (TMPD photo-oxidized). Double flash experiments show that maximal utilization of a second flash for phosphorylation (as with dye photo-oxidation) requires complete recovery of the initial absorbance change induced by the first flash. These experiments show that the rapid recovery of photochemical reaction centers induced by both TMPD and PMS allows accumulation of intermediates involved in photophosphorylation during repetitive excitation. The dyes thus increase the flash yield of ATP obtained with 2-msec flashes. The rate of phosphorylation in darkness following a 2-msec flash also is increased by the dyes.