PIGMENT ORGANIZATION IN THE LIGHT-HARVESTING CHLOROPHYLL-a/b PROTEIN COMPLEX OF LETTUCE CHLOROPLASTS. EVIDENCE OBTAINED FROM PROTECTION OF THE CHLOROPHYLLS AGAINST PROTON ATTACK and FROM EXCITATION ENERGY TRANSFER

Abstract— The light-harvesting Chl-a/b protein complex (LHC) of Lactuca sativa L. was examined for pigment content, excitation energy transfer and behavior under acidic conditions:
(1) Lettuce LHC contains Chl-a, Chl-b and xanthophylls (lutein, neoxanthin, lactucaxanthin, viola-xanthin) at a molar ratio of 6:4:3; their contribution to the absorbance of the LHC between 390 and 530 nm is estimated to be about 31% (Chl-a), 26% (Chl-h) and 43% (xanthophylls).
(2) Energy transfer from xanthophylls and Chl-fe to Chl-a takes place at 100% transfer efficiency.
(3) LHC exhibits an unusual acid stability: in contrast to complexes of photosystem I or II, LHC-bound chlorophylls are not converted to phaeophytin and LHC apoprotein is not denatured at pH 1.5; also, energy transfer is maintained.
(4) Pronase or trypsin treatment do not affect acid stability and energy transfer.
(5) Treatments that break down acid stability (heat, urea or TritonX–100) also inhibit energy transfer.
The coincidental breakdown of energy transfer and acid stability points at one underlying process, namely, the breakdown of a structure that enables protection of chlorophylls from proton attack and close contiguity of xanthophylls and chlorophylls as required for energy transfer. Dense packing of xanthophylls and chlorophylls within lipophilic crevices of the LHC is suggested.     

EVIDENCE FOR A HETEROGENOUS ORGANIZATION OF VIOLAXANTHIN IN THYLAKOID MEMBRANES

Abstract Two functionally different species of violaxanthin have been observed in thylakoid membranes, one that can be de-epoxidised to zeaxanthin under light and one not available for light-induced zeaxanthin formation (Siefermann, D. and H. Y. Yamamoto, 1974, Biochim. Biophys. Acta 357 , 144–150). Here the distribution of available and unavailable violaxanthin is examined between membrane subfractions obtained from Triton X-100 solubilized spinach thylakoids by isoelectric focusing: (1) Only 40% of the available violaxanthin is detected in isolated Chl-proteins, while the residual 60% occur in a fraction of'free'pigments; (2) Almost 80% of the unavailable violaxanthin is recovered from the light-harvesting Chl a/b -protein complex (36%) and from photochemically active complexes containing photosystem I (20%) or photosystem II (20%). The results suggest a heterogenous organization of available and unavailable violaxanthin in thylakoid membranes.     

Evidence for a pterin-derivative associated with the molybdenum cofactor of Neurospora crassa nitrate reductase

Abstract— Following the method of Johnson and Rajagopalan (1982) for obtaining Form B of molybdopterin cofactors, we observed a prominent fluorescence band at480–482 nm in purified NR of Neurospora crassa mutant albino-band after boiling the enzyme at acidic pH and readjusting the sample to alkaline pH. This fluorescence band is maximally excited at 410 nm and maximally emitting at pH 11 (“F-480_pH11”); at pH4–7 only a featureless fluorescence band of low intensity remains (“F-480_pH5”). The fluorescence ΔF-480 = F-480_pH11 - F-480_pHS is examined here. ΔF-480 is associated specifically with NADPH-dependent and MVH-dependent nitrate reduction activities and with cytochrome b-557 absorption. In a protease-digested preparation lacking NADPH-dependent NR activity, ΔF-480 is associated with MVH-dependent nitrate reduction. The ΔF-480 signal is followed during the course of purification of NR. Its size increases with increasing purity of the enzyme. In partially purified NR preparations and especially in aqueous extracts from mycelia of N. crassa, a second, strong fluorescence signal with a pH-dependent emission maximum at around 450 nm (maximally excited at350–370 nm) was found beside ΔF-480. This “unspecific” signal was lost during NR purification. A procedure is developed to demonstrate AF-480 also in presence of the unspecific (350-370/450 nm) signal as well as flavins. We deduce that the ΔF-480 component is part of the Mo cofactor of N. crassa NR and that the signal is caused by a pterin derivative. From calculations of total content of the AF-480 component in mycelia it is likely that in vivo it is shared also by other enzymes.