Noriaki Kusumoto

Function of the Reaction Center of Green Sulfur Bacteria

The reaction center (RC) of green sulfur bacteria belongs to the Fe‐S type RC, as do the photosystem I of oxygenic photosynthetic organisms and the RC of heliobacteria. The core parts of the green sulfur bacterial and the heliobacterial RC are assumed to be homodimeric, in contrast to those of purple bacteria, photosystem I and photosystem II. This paper describes recent advances in the study of the function of the green sulfur bacterial RC.

Purification of ferredoxins and their reaction with purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Four ferredoxin (Fd) fractions, namely, FdA-D were purified from the green sulfur bacterium Chlorobium tepidum. Their absorption spectra are typical of 2[4Fe-4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. The A(385)/A(280) ratios of the purified Fds were 0.76-0.80. Analysis of the N-terminal amino acid sequences of these Fds (15-25 residues) revealed that those of FdA and FdB completely agree with those deduced from the genes, fdx3 and fdx2, respectively, found in this bacterium (Chung and Bryant, personal communication). The N-terminal amino acid sequences of FdC and FdD (15 residues) were identical, and agree with that deduced from the gene fdx1 (Chung and Bryant, personal communication). The A(385) values of these Fds were unchanged when they were stored for a month at -80 degrees C under aerobic conditions and decreased by 10-15% when they were stored for 6 days at 4 degrees C under aerobic conditions, indicating that they are not extremely unstable. In the presence of Fd-NADP(+) reductase from spinach, and a purified reaction center (RC) preparation from C. tepidum composed of five kinds of polypeptides, these Fds supported the photoreduction of NADP(+) at room temperature with the following K(m) and V(max) (in micromol NADP(+) micromol BChl a(-1) h(-1)): FdA, 2.0 microm and 258; FdB, 0.49 microM and 304; FdC, 1.13 microM and 226; FdD, 0.5 microM and 242; spinach Fd, 0.54 microM and 183. The V(max) value of FdB was more than twice that previously reported for purified RC preparations from green sulfur bacteria.

Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c551 and an Fe-S center (CFA/CFB) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CFA/CFB) which decayed with a t1/2 of ∼400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em −360 mV) and methyl viologen (−440 mV), and less effectively by triquat (−540 mV). The difference spectrum of Cyt c had negative peaks at 551, ∼520 and ∼420 nm, with a positive rise at ∼440 to ∼500 nm. The difference spectrum of CFA/CFB resembled P430 of PSI, and had a broad negative peak at 430∼435 nm.

Reduction of ferredoxins by photosynthetic reaction center complex from the green sulfur bacterium Chlorobium tepidum

Green sulfur bacterial reaction centers (RCs) belong to an iron-sulfur type RC, and can photoreduce low potential iron-sulfur protein, ferredoxin, as does PSI. From the thermofilic green sulfur bacterium, C. tepidum, four 2[4Fe-4S] type Fd fractions were purified. The amount of each Fd fractions varied depending upon culture conditions. Absorbances at 385nm of these Fds decreased by 10-15% in 6 days at 4oC in aerobic condition, indicating that these Fds were rather stable in contrast with previous reports. Two of these Fds? N-terminal amino acid sequences (15 residues) were completely identical. All of these Fds? N-terminal amino acid sequences (15-25 residues) completely agreed to those deduced from the three fdx genes (Chung S., and Bryant D.A.). These Fds are photoreducible by purified RC from this bacterium, and support NADP+ photoreduction with spinach ferredoxin-NADP+ reductase. The Vmax and Km of electron transfer rates in the above reaction were significantly different among Fds.

Activities and Kinetics of Electron Transfer in a Reaction Center Complex from the Green Sulfur Bacterium Chlorobium Tepidum

The reaction center (RC) of green sulfur bacteria (PS-C) belongs to a Type 1 photosystem as PSI and RC of heliobacteria (PS-H). These RCs have Fe-S center(s) as electron acceptors, and their core polypeptides show amino acid sequence similarities among each other. However, the RC core polypeptides of PS-C and PS-H are assumed to be homodimeric in contrast with PSI. The sequence and kinetics of electron transfer in PSC as well as to and from PS-C are only partially elucidated (1,2). PS-C can directly photoreduce ferredoxin (Fd) in contrast with purple bacterial RC and we report here some of the results of kinetic studies. Kjwr and Scheller (3) found that purified PS-C from Chlorobium vibriofome photoreduced NADP+ in the presence of Fd from Clostridium and FNR from spinach. We purified several Fds from Chlorobium tepidum and studied their activities as electron mediators in NADP+ photoreduction. In PSI, phylloquinone functions as an electron mediator (A1) between A0 and Fx (A2). In heliobacterial membranes, procedures which were assumed to extract quinones did not result in significant changes in the yield of stable charge separation, suggesting that menaquinone is not an essential participant in the electron acceptor chain (4). More recently, Brettel et al. (5) concluded from time-resolved flash spectroscopy in ns time range and from photovoltage measurements of heliobacterial membranes that quinone is not functioning as an electron acceptor in PS-H. In PS-C, there have been controversies over the functioning of quinone as an electron acceptor (reviewed in (1, 2)). In order to answer these questions, we studied electron transfer kinetics in purified PS-C in ns-ms time range by flash absorption spectroscopy. We also measured the reduction rate of photooxidized P840 by Cyt c551 bound to PS-C.

The Sites of Photoinhibition Around Photosystem I in Chloroplasts

Illumination of chloroplasts with strong light brings about photoinhibition of photosynthetic electron transport around PSI as well as around PSII (1). In contrast with photoinhibition of PSII, photoinhibition of PSI usually requires the presence of O2 (2). We have found that the main cause of photoinhibition of PSI under aerobic conditions is the destruction of three Fe-S centers by some species of active oxygen produced by illuminated broken chloroplasts (3).