ABSTRACT
Photosynthesis converts solar energy to chemical energy using chlorophylls (Chls). In a late stage of biosynthesis of Chls, dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR), a nitrogenase-like enzyme, reduces the C17 = C18 double bond of Pchlide and drastically changes the spectral properties suitable for photosynthesis forming the parental chlorin ring for Chl a. We previously proposed that the spatial arrangement of the proton donors determines the stereospecificity of the Pchlide reduction based on the recently resolved structure of the DPOR catalytic component, NB-protein. However, it was not clear how the two-electron and two-proton transfer events are coordinated in the reaction. In this study, we demonstrate that DPOR initiates a single electron transfer reaction from a [4Fe-4S]-cluster (NB-cluster) to Pchlide, generating Pchlide anion radicals followed by a single proton transfer, and then, further electron/proton transfer steps transform the anion radicals into chlorophyllide (Chlide). Thus, DPOR is a unique iron-sulphur enzyme to form substrate radicals followed by sequential proton- and electron-transfer steps with the protein folding very similar to that of nitrogenase. This novel radical-mediated reaction supports the biosynthesis of Chl in a wide variety of photosynthetic organisms.
Subject(s)
Bacteriochlorophyll A/chemical synthesis , Iron/chemistry , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Protochlorophyllide/chemistry , Sulfur/chemistry , Bacteriochlorophyll A/biosynthesis , Enzyme Activation/radiation effects , Free Radicals , Iron/radiation effects , Light , Oxidoreductases Acting on CH-CH Group Donors/radiation effects , Photosynthesis/physiology , Photosynthesis/radiation effects , Protochlorophyllide/radiation effects , Substrate Specificity , Sulfur/radiation effectsABSTRACT
A series of new bacteriochlorins was synthesized using 13(2)-oxo-bacteriopyropheophorbide a (derived from bacteriochlorophyll a) as a starting material, which on reacting with o-phenylenediamine and 1,10-diaminonaphthalene afforded highly conjugated annulated bacteriochlorins with fused quinoxaline, benzimidazole, and perimidine rings, respectively. The absorption spectra of these novel bacteriochlorins demonstrated remarkably red-shifted intense Q(y) absorption bands observed in the range of 816-850 nm with high molar extinction coefficients (89,900-136,800). Treatment of 13(2)-oxo-bacteriopyropheophorbide a methyl ester with diazomethane resulted in the formation of bacterioverdins containing a fused six-membered methoxy-substituted cyclohexenone (verdin) as an isomeric mixture. The pure isomers which exhibit long-wavelength absorptions in the near-IR region (865-890 nm) are highly stable at room temperature with high reactivity with O(2) at the triplet photoexcited state and favorable redox potential and could be potential candidates for use as photosensitizers in photodynamic therapy (PDT).
Subject(s)
Bacteriochlorophyll A/chemistry , Bacteriochlorophyll A/chemical synthesis , Porphyrins/chemistry , Porphyrins/chemical synthesis , Absorption , Electrochemistry , Molecular Structure , Photochemistry , Photochemotherapy , Photosensitizing Agents , Quantum Theory , Spectroscopy, Near-InfraredABSTRACT
Among the photosensitizers investigated, both ring-D and ring-B reduced chlorins containing the m-iodobenzyloxyethyl group at position-3 and a carboxylic acid functionality at position-17(2) showed the highest uptake by tumor cells and light-dependent photoreaction that correlated with maximal tumor-imaging [positron emission tomography (PET) and fluorescence] and long-term photodynamic therapy (PDT) efficacy in BALB/c mice bearing Colon26 tumors. However, among the ring-D reduced compounds, the isomer containing the 1'-m-iobenzyloxyethyl group at position-3 was more effective than the corresponding 8-(1'-m-iodobenzyloxyethyl) derivative. All photosensitizers showed maximum uptake by tumor tissue 24 h after injection, and the tumors exposed with light at low fluence and fluence rates (128 J/cm(2), 14 mW/cm(2)) produced significantly enhanced tumor eradication than those exposed at higher fluence and fluence rate (135 J/cm(2), 75 mW/cm(2)). Interestingly, dose-dependent cellular uptake of the compounds and light-dependent STAT3 dimerization have emerged as sensitive rapid indicators for PDT efficacy in vitro and in vivo and could be used as in vitro/in vivo biomarkers for evaluating and optimizing the in vivo treatment parameters of the existing and new PDT candidates.
Subject(s)
Bacteriochlorophyll A/chemical synthesis , Chlorophyll/chemical synthesis , Photosensitizing Agents/chemical synthesis , Radiopharmaceuticals/chemical synthesis , STAT3 Transcription Factor/metabolism , Animals , Bacteriochlorophyll A/chemistry , Bacteriochlorophyll A/pharmacology , Cell Line, Tumor , Chlorophyll/chemistry , Chlorophyll/pharmacology , Chlorophyll A , Humans , Iodine Radioisotopes , Isomerism , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Neoplasms, Experimental/diagnostic imaging , Neoplasms, Experimental/drug therapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Positron-Emission Tomography , Protein Multimerization , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/pharmacology , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
The 3-acetyl groups of synthetic methyl pyropheophorbides were rotated around the 3-3(1) bond and the rotational conformers were obtained in a dichloromethane solution of 20-bromo- and methyl-substituted compounds, based on their electronic and vibrational absorption spectra. Such a rotational deviation of the 3-acetyl group from the cyclic tetrapyrrole plane induced less pi-conjugation to affect the redmost Q(y) band, which has been observed in natural photosynthetic antenna systems, bacteriochlorophyll-a molecules in oligopeptides.
