EPR and optical spectroscopic properites of the electron carrier intermediate between the reaction center bacteriochlorophylls and the primary acceptor in Chromatium vinosum

1. A reaction center-cytochrome c complex has been isolated from Chromatium vinosum which is capable of normal photochemistry and light-activated rapid cytochrome c₅₅₃ and c₅₅₅ oxidation, but which has no antenna bacteriochlorophyll. As is found in whole cells, ferrocytochrome c₅₅₃ is oxidized irreversibly in milliseconds by light at 7 K.2. Room temperature redox potentiometry in combination with EPR analysis at 7 K, of cytochrome c₅₅₃ and the reaction center bacteriochlorophyll dimer (BChl)₂ absorbing at 883 nm yields identical results to those previously reported using optical analytical techniques at 77 K. It shows directly that two cytochrome c₅₅₃ hemes are are equivalent with respect to the light induced (BChl)₂ At 7 K, only one heme can be rapidly oxidized in the light, commensurate with the electron capacity of the primary acceptor (quinone-iron) being unity.3. Prior chemical reduction of the quinone-iron followed by illumination at 200K, however, leads to the slow ( ) oxidation of one cytochrome c₅₅₃ heme, with what appears to be concommitant reduction of one of the two bacteriophytins (BPh) of the reaction center as shown by bleaching of the 760 nm band, a broad absorbance increase at approx. 650 nm and a bleaching at 543 nm. The 800 nm absorbing bacteriochlorophyll is also involved since there is also bleaching at 595 and 800 nm; at the latter wave-length the remaining unbleached band appears to shift significantly to the blue. No redox changes in the 883 absorbing bacteriochlorophyll dimer are seen during or after illumination under these conditions. The reduced part of the state represents what is considered to be the reduced form of the electron carrier (I) which acts as an intermediate between the bacteriochlorophyll dimer and quinoneiron. The state (oxidized ) relaxes in the dark at 200 K in approx. 20 min but below 77 K it is trapped on a days time scale.4. EPR analysis of the state trapped as described above reveals that one heme equivalent of cytochrome becomes oxidized for the generation of the state, a result in agreement with the optical data. Two prominent signals are associated with the trapped state in the g = 2 region, which can be easily resolved with temperature and microwave power saturation: one has a line width of 15 g and is centered at g = 2.003; the other, which is the major signal, is also a radical centered at g = 2.003 but is split by 60 G and behaves as though it were an organic free-radical spin-coupled with another paramagnetic center absorbing at higher magnetic field values; this high field partner could be the iron-quinone of the primary acceptor. The identity of two signals associated with I is consistent with the idea that the reduced intermediary carrier is not simply BPh but also involves a second radical, perhaps the 800 nm bacteriochlorophylls in the reduced state. As such, the single electron would be shared in some way, and it is probable that one of these centers will be very close to the paramagnetism of the iron-quinone. Alternatively, it is possible that the electron only occupies BPh (the optical changes associated with the 800 nm bacteriochlorophyll occurring on a secondary basis) and that some of the BPh population of the trapped state is not close enough to interact with the quinone-iron.5. Light-induced triplet state formation is dramatically diminished in material in which I as well as the quinone-iron is reduced before illumination. This supports the idea that with quinone-iron alone reduced before illumination, triplet formation requires light activated electron transfer from the bacteriochlorophyll dimer to I (not possible if I is already reduced) and that the triplet is formed by the return of the electron from I to (BChl)₂.6. Results indicate that although the two cytochrome c₅₅₃ hemes may be equivalent at the point of activation, once one has become oxidized the other becomes less competent for oxidation by the (BChl)₂.