Electron paramagnetic resonance studies of photosynthetic electron transport: Photoreduction of ferredoxin and membrane-bound iron-sulfur centers

Abstract

Electron paramagnetic resonance spectrometry was used to investigate, at physiological temperatures, lightinduced electron transport from membrane-bound iron-sulfur components (bound ferredoxin) to soluble ferredoxin and NADP+ in membrane fragments (from the blue-green alga, Nostoc muscorum) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NADP+. Illumination at 200 resulted in the photoreduction of membrane-bound iron-sulfur centers A and B. Photoreduction by water gave electron paramagnetic resonance signals of both centers A and B; photoreduction by DCIPH2 was found to generate a strong electron paramagnetic resonance signal of only center B.When water was the reductant, the addition and photoreduction of soluble ferredoxin generated additional signals characteristic of soluble ferredoxin without causing a decrease in the amplitude of the signals due to centers A and B. The further addition of NADP+ (and its photoreduction) greatly diminished signals due to the bound iron-sulfur centers and to soluble ferredoxin. An outflow of electrons from center B to soluble ferredoxin and NADP+ was particularly pronounced when DCIPH2 was the reductant. These observations provide the first evidence for a light-induced electron transport between membrane-bound iron-sulfur centers and ferredoxin-NADP+. The relationship of these observations to current concepts of photosynthetic electron transport is discussed.The now well-documented broad importance of iron-sulfur proteins to photosynthesis (reviewed in ref. 1) became apparent in two stages. First, when photoreduced chloroplast ferredoxin was found to be both the electron donor (with a midpoint potential of -420 mV) for NADP+ reduction (2-4) (17)(18)(19)(20)(21)(22) assigned the role of primary electron acceptor in photosystem I to center A which, unlike the more electronegative center B, undergoes a photoreduction at cryogenic temperatures concurrently with the photooxidation of P700. There is as yet no evidence bearing on the role of center B in photosystem I although it appears that center B may be interacting with center A under certain experimental conditions. Aside from the identity of the primary electron acceptor of photosystem I, there is a general assumption, unsupported so far by any experimental evidence, of electron transfer between the bound iron-sulfur centers of photosystem I and soluble ferredoxin. The reduction of centers A and B was investigated under drastic experimental conditions-i.e., under illumination at cryogenic temperatures (<77 K) and in the presence of a strong nonphysiological reductant (dithionite)-that are not compatible with the reduction of ferredoxin and NADP+ either by the physiological reductant water or by substitute electron donors such as the reduced dye, 2,6-dichlorophenolindophenol (DCIPH2). Photoreduction

Membrane-bound iron-sulfur centers in photosynthetic systems

“…The recent proposal of Arnon et a1. [15] has some similarities to that of Bolton in that it proposes that the bound iron-sulfur centers are functioning in different electron transfer processes. As has been previously pOinted out, however, the conclusions in the latter case are not based on a correct interpretation of the EPR data and the results can also be explained in terms of both Center A and B interacting with soluble ferredoxin and NADP regardless of the electron donor system used.…”

“…In contrast to theseresuits, one detergent, sodium dodecyl sulfate (SDS), has unusual effeCts in terms of the Photosystem I reaction center complex,aridthese will be discussed in greater detail in a subsequent section. ,Bound iron-sulfur centers' have also been observed in a number of different preparations •froinalgal species, including green algae [14], and blue-green algae [14,15], as wei) as in 'enriched Phot'osystem I preparations from such organisms ' [13]:. In contrast to'this \\,jde"spread'distribution, ,no bound iron-sulfur centers• are ,observed in a highly purified Photosysiem U r~actioi:t'center'complex prepared from spinach chloroplasts with Triton the reaction center becomes more enriched in such preparations (based on P-700 content), the enrichment of the bound iron-sulfur center shows a parallel increase.…”

“…A recent report has described the photoreduction of Center A and B in oxygenevolving fragments from a blue-green alga [15]. Attempts were made to study shifts in steady-state levels of these centers after the addition of electron acceptor systems, but the conclusions drawn from this work are not correct because of errors in interpreting the g-values in the reduced EPR spectra.…”

“…8 that the g = 1.89 resonance line does not originate solely from Center B but has a contribution from Center A (the shifted g = 1.86 resonance line) on complete reduction. Arnon et al [15] have concluded from their studies that only Center B is involved in the photoreduction of NADP+ with reduced dichlorophenolindophenol as the electron donor. This conclusion is based on an observed shift in the steady-state level of the g = 1.89 resonance line on the addition of an electron acceptor.…”