Reactions of Isocytochrome c₂ in the Photosynthetic Electron Transfer Chain of Rhodobacter sphaeroides

Abstract

Rhodobacter sphaeroides strains lacking cytochrome c2 (cyt c2), the normal electron donor to P870+ in light-oxidized reaction center (RC) complexes, are unable to grow photosynthetically. However, spd mutations that suppress the photosynthetic deficiency of cyt c2 mutants elevate levels of the cyt c2 isoform, isocyt c2. We monitored photosynthetic electron transfer in whole cells, in chromatophores, and with purified components to ascertain if and how isocyt c2 reduced light-oxidized RC complexes. These studies revealed that several fundamental aspects of photosynthetic electron transfer were similar in strains that use isocyt c2 and wild-type cells. For example, P870+ reduction accompanied cytochrome c oxidation. In addition, photosynthetic electron transfer was blocked by the well-known cyt bc1 complex inhibitors antimycin and myxothiazol. However, even at the increased isocyt c2 levels present in these strains (approximately 40% that of cyt c2 in wild-type cells), there was little, if any, of the rapid (< 5 microns) electron transfer to P870+ that is characteristic of cytochromes bound to RC complexes at the time of the light flash. Thus, it appears that isocyt c2 function limits the in vivo rate of P870+ reduction. Indeed, at low ionic strength in vitro, the apparent affinity of isocyt c2 for RC complexes (KD approximately 40 microM) is significantly lower than that of cyt c2 (KD approximately 1.0 microM). This reduced affinity does not appear to result from an altered mode of RC binding by isocyt c2 since electrostatic interactions make similar overall contributions to the binding of both cyt c2 and isocyt c2 to this membrane-bound redox partner. Thus, sequence, structural, or local conformational differences between cyt c2 and isocyt c2 significantly alter their apparent affinities for this physiologically relevant redox partner.

The membrane-attached electron carrier cytochrome c y from Rhodobacter sphaeroides is functional in respiratory but not in photosynthetic electron transfer

“…This has been proposed to be the case for acidic isocytochrome c 2 (pI ϭ 4.87) of R. sphaeroides, which has Ϸ40-fold lower affinity for the reaction center in vitro compared with Cyt c 2 Rs (pI ϭ 7.01). It is thought that the negative charge of isocytochrome c 2 brings about its lowered affinity toward the reaction center by changing the orientation of the dipole moment at the isocytochrome c 2 docking surface (35). In any event, a relatively low affinity for binding to the reaction center of the Cyt c domain of the Cyt c y Rs may explain, at least partially, the Ps incapability of Cyt c y Rs and suggests that its interactions with the Cyt c oxidase may be different than those with the reaction center.…”

The Energy Landscape for Ubihydroquinone Oxidation at the Qo Site of the bc 1 Complex inRhodobacter sphaeroides

“…The kinetic spectrophotometer used was of conventional single-beam design, as described (30). The cuvette was stirred from the bottom using a stirring bar, and an electromagnetic driver was constructed in-house.…”

Unbinding of Oxidized Cytochrome c from Photosynthetic Reaction Center of Rhodobacter sphaeroides Is the Bottleneck of Fast Turnover

“…The RC-cyt c system displays kinetically distinct phases. The fast phase [t 1/2 ∼ 1 µs (4-7)] arises from "proximal" (bound) cytochrome at the time of the flash, and the slower phases [t 1/2 > 200 µs (8)(9)(10)(11)(12)(13)(14); t 1/2 ∼ 100 µs (6); t 1/2 ∼ 65 µs (15); and t 1/2 ∼ 55 µs (4)] are attributed to the "off" (unbound) or "distal" (bound) states that require association or reorientation reactions prior to electron transfer. Measurements on optical linear dichroism (16,17), EPR (17), viscosity (13), and site-directed (L162) mutant of the RC (6) indicated that the proximal and distal bound cytochromes are physically distinct, although some authors did not find evidence for a distal site (7,(18)(19)(20).…”