The kinetics of photo-induced electron transfer from high-potential iron-sulfur protein (HiPIP) to the photosynthetic reaction center (RC) of the purple phototroph Rhodoferaxfermentans were studied. The rapid photooxidation of heme c-556 belonging to RC is followed, in the presence of HiPIP, by a slower reduction having a second-order rate constant of4.8 x 107 M -l s -1. The limiting value of k.bs at high HiPIP concentration is 95 s-1. The amplitude of this slow process decreases with increasing HiPIP concentration. The amplitude of a faster phase, observed at 556 and 425 nm and involving heme c-556 reduction, increases proportionately. The rate constant of this fast phase, determined at 425 and 556 nm, is -3 x 105 s-1. This value is not dependent on HiPIP concentration, indicating that it is related to a first-order process. These observations are interpreted as evidence for the formation of a HiPIP-RC complex prior to the excitation flash, having a dissociation constant of -2.5 ,uM. The fast phase is absent at high ionic strength, indicating that the complex involves mainly electrostatic interactions. The ionic strength dependence of kobs for the slow phase yields a second-order rate constant at infinite ionic strength of 5.4 x 106 M-t.s-I and an electrostatic interaction energy of -2.1 kcal/mol (1 cal = 4.184 J). We conclude that Rhodoferax fermentans HiPIP is a very effective electron donor to the photosynthetic RC.Anoxygenic phototrophic bacteria contain two membranebound components that are essential for energy production, the photosynthetic reaction center (RC) and the cytochrome bc1 complex. Two types of RC have been structurally characterized: the RC from Rhodobacter sphaeroides is made of three subunits (1, 2), while, in addition to these, RC from Rhodopseudomonas viridis contains a fourth tetraheme cytochrome c subunit (2, 3). InRps. viridis the four heme groups have a bands at 559, 552, 556, and 554 nm, and reduction potentials of +380 mV, +20 mV, +310 mV, and -60 mV, respectively (4). The four hemes are arranged in an almost linear fashion, (2,5,6) and are aligned in the sequence P/c-559/c-552/c-556/c-554, where P indicates the bacteriochlorophyll special pair (4, 7). The highest potential heme (c-559) is oxidized the most rapidly (t/2 -300 ns) by the photooxidized P (P+), followed by a slower oxidation of heme c-556 (ti/2 2 ,us) (4,8,9). The quinol generated by RC photooxidation is utilized by the bc1 complex to reduce cytochrome c2 (10), a soluble protein closely related to mitochondrial cytochrome c (11,12). Cytochrome c2 then reduces the photooxidized RC, closing the photocycle. However, although cytochrome c2 reacts directly with P+ in Rb. sphaeroides (13-21), in Rps. viridis, it reacts with the tetraheme subunit (22-24). Only about half of the purple bacterial species described so far contain cytochrome c2 (25), and it is not known how the remaining species carry out electron transfer between the bc1 and RC complexes.The RC from the purple nonsulfur bacterium Rhodoferax fermentans (26)...

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The capability of high potential iron-sulfur proteins (HiPIPs) and soluble cytochromes to shuttle electrons between the bc1 complex and the terminal oxidase in aerobically grown cells of Rhodoferax fermentans and Rhodospirillum salinarum, two facultative phototrophs, was evaluated. In Rs. salinarum, HiPIP and a c-type cytochrome (alpha-band at 550 nm, Em,7=+290 mV) are both involved in the electron transfer step from the bc1 complex to the terminal oxidase. Kinetic studies indicate that cytochrome c550 is more efficient than HiPIP in oxidizing the bc1 complex, and that HiPIP is a more efficient reductant of the terminal oxidase as compared to cytochrome c550. Rs. salinarum cells contain an additional c-type cytochrome (asymmetric alpha-band at 556 nm, Em,7=+180 mV) which is able to reduce the terminal oxidase, but unable to oxidize the bc1 complex. c-type cytochromes could not be isolated from Rf. fermentans, in which HiPIP, the most abundant soluble electron carrier, is reduced by the bc1 complex (zero-order kinetics) and oxidized by the terminal oxidase (first-order kinetics), respectively. These data, taken together, indicate for the first time that HiPIPs play a significant role in bacterial respiratory electron transfer.

The legend to Figure 1 1 should read as follows:The stability of Mitochondria-containing AOT micelles against temperature and water content (Wo). The AOT concentration is constant (100 mM). The water-in-oil microemulsion contained 97 micrograms of mitochondria1 proteins/mL of micellar solution.The legend to

The water-in-oil microemulsion system bis(2 ethyl-hexyl-sodium-succinate (AOT)/isooctane/water is able to solubilize soybean nodules mitrochrondria. Transparent and thermodynamically stable hydrocarbon solutions are obtained, which can be assayed for mitochondrial activity just as aqueous solutions. Malate dehydrogenase (MDH) activity was measured in vivo and gave in reverse micelles very similar results as in water. However the kinetic behavior of this reaction in AOT/isooctane reverse micelles shows some differences with respect to water. Mitochondria in reverse AOT micelles are able to retain about 70% of their initial MDH activity after three days. Mitochondria can be back-transferred from reverse micelles to water and show respiratory activity almost identical to the native organelles. Electron microscopy studies show that the dimensions of mitochondria back-transferred into water from AOT micelles are comparable to the dimensions of the native organelles.