Chemical tools to study photosynthesis

Figure 1. Schematic representation of the electron transfer chain with commonly used chemical tools. Figure adapted from: http://jonnield.com/en/science/psII_images.html   

DCMU (3-(3′,4′-dichlorophenyl)-1,1-dimethylurea)

DCMU displaces the secondary quinone acceptor, QB, from its binding site at the D1 protein of Photosystem II (PSII) [1], [2]. DCMU is not redox active and prevents the re-oxidation of QA− by forward electron transport.[3]

DBMIB (dibromothymoquinone) 

DBMIB inhibits the reoxidation of plastoquinol by binding to the cytochrome b6/f complex [1]. DBMIB is an artificial quinone introduced in 1970 by Trebst et al. [2] and Böhme et al. [3] as an inhibitor of photosynthetic electron transport and an antagonist of PQ. It was shown that cyt b6/f can accept only one electron from DBMIB. As a semiquinone it remains tightly bound to the cyt b6/f complex preventing in this way the reoxidation of other PQH2 molecules by cyt b6/f[4].

MV (methylviologen; paraquat) 

MV accepts electrons from the FeS clusters of PSI and it allows electrons to bypass the block that is transiently imposed by ferredoxin-NADP+-reductase (FNR) (inactive in dark-adapted chloroplasts) [1]. MV is thought to be a very effective electron acceptor that competes strongly with ferredoxin for electrons from the FeS clusters of PSI and, as a consequence, strongly suppresses cyclic electron transfer around PSI [2], [3].

MV is not a direct inhibitors of the electron transport system, but rather a drain off electrons from photosystem I, preventing NADP reduction [4].

Sulfo-DSPD (disulfodisalicylidenepropanediamine) 

Sulfo-DSPD has been shown to inhibit ferredoxin-dependent reactions with both chloroplast and membrane-free systems [1,2,3]. Its site of inhibition has been placed before ferredoxin reduction in the electron transport sequence [2] or at ferredoxin itself [3]. 

Sulfo-DSPD inhibits at the acceptor side of photosystem I (lipophilic DSPD does not), but because of its very hydrophilic nature, it is active only in exposed thylakoid membrane preparations [4]. 

AA (Antimycin A3)

AA inhibits PGR5–PGRL1-dependent PSI cyclic electron transport [1]. Chloroplast NDH also accepts electrons from ferredoxin (Fd) but is resistant to AA [2]. AA was originally discovered to inhibit respiratory electron transport by binding to the Qi site of the cytochrome (Cyt) bc1 complex [3]. However, AA does not bind to the corresponding site of the Cyt b6f complex in chloroplasts. AA was shown to inhibit electron transport from recombinant PGRL1 to the plastoquinone (PQ) analog 2,6-dimethyl-p-benzoquinone in vitro [4], and PGR5 may function in the Fd-dependent reduction of PGRL1 in vivo [5]. Consistent with these results, a single amino acid alteration in PGR5 confers resistance of PSI cyclic electron transport to AA [6]. AA most likely inhibits the function of the PGR5–PGRL1 protein complex, although the exact mode of inhibition is still unclear [1]. 

Asc (Ascorbate, vitamin C)

Asc has been shown to support DCMU-sensitive photoreduction of NADP+ in thylakoids isolated from heat-treated Euglena gracilis cells [1] and it has also been shown to donate electrons to PSII in tris(hydroxymethyl)aminomethane (Tris)-washed thylakoids [2]. Asc donates electrons to TyrZ+ as shown by electron paramagnetic resonance [3] and thermoluminescence measurements [4, 5]. When PSII is inhibited by DCMU in isolated thylakoid membranes, Asc can act as an electron donor to PSI [6].

DCPIP (Dichlorophenol-indophenol) 

DAD (Diaminodurene = 2,3,5,6-tetramethyl-pphenylenediamine)

TMPD (N-tetramethyl-p-phenylenediamine)

Donor systems for photosystem I such as DCPIP, DAD or TMPD, kept reduced by ascorbate, are not inhibited by either DCMU or DBMIB. They are coupled to ATP formation [1]. 

DPC (Diphenylcarbazide) 

Donor systems for photosystem I such as DCPIP, DAD or TMPD, kept reduced by ascorbate, are not inhibited by either DCMU or DBMIB. They are coupled to ATP formation [1]. 

Hydroxylamine (NH2OH) 

CCCP (carbonylcyanide-phenylhyrazones)

Hydroxylamine and carbonylcyanide-phenylhyrazones (CCCP) act on the donor side of photosystem II both as inhibitor and donor. [1]. Hydroxylamine action on PSII occurs in two steps: an initial reversible reduction of manganese by two electrons at low concentrations (≤5 NH2OH / PSII) followed by, at higher concentrations, further reduction that is irreversible due to the release of 3 out of 4 Mn/PSII [2, 3]. Hydroxylamine can also be used for manganese-depletion accomplished by incubating thawed Ca-depleted PSII membranes (0.5 mg Chl/ml) for 5 min at 5 °C in the dark with 1 mM hydroxylamine [4, 5]. 

CCCP is oxidized by the photosystem II donor side and is reduced by the plastoquinon pool [6]. CCCP is additionally an effective protonophone which can be used to disipathe the DeltapH [7].

mercuric chloride (HgCl2)

Incubation of chloroplasts with HgCl2 at a molar ratio of HgCl2 to chlorophyll of about unity, induced a complete inhibition of the methylviologen Hill reaction, as well as methylviologen photoreduction with reduced DCPIP as electron donor. [1]. 

ionophores


Nigericin

Nigericin dissipates chloroplast thylakoid membrane proton gradient by transferring H+ ions into the lumen [1]. Nigericin is a linear molecule with heterocyclic oxygen-containing rings together with a hydroxyl group. It catalyses the overall electroneutral exchange of K+ for H+ [2].


FCCP (carbonylcyanide-p-(trifluoromethoxy) phenylhydrazone) 

The protonophore FCCP has been shown to be able to decrease the cellular ATP content and to increase the NADPH concentration [1, 2, 3]. FCCP is the most commonly employed example of a protonophore [4].


Valinomycin

Exposure of biological membranes to the ionophore valinomycin selectively increases K+ conductance and allows for rapid electrochemical gradient-driven K+ fluxes. [1, 2]

Good learning resource from Larry Orr and Govindjee:

https://www.life.illinois.edu/govindjee/photoweb/individual.html