Study of the high-potential iron sulfur protein in Halorhodospira halophila confirms that it is distinct from cytochrome c as electron carrier

Abstract

The role of high-potential iron sulfur protein (HiPIP) in donating electrons to the photosynthetic reaction center in the halophilic gamma-proteobacterium Halorhodospira halophila was studied by EPR and time-resolved optical spectroscopy. A tight complex between HiPIP and the reaction center was observed. The EPR spectrum of HiPIP in this complex was drastically different from that of the purified protein and provides an analytical tool for the detection and characterization of the complexed form in samples ranging from whole cells to partially purified protein. The bound HiPIP was identified as iso-HiPIP II. Its Em value at pH 7 in the form bound to the reaction center was approximately 100 mV higher (+140 +/- 20 mV) than that of the purified protein. EPR on oriented samples showed HiPIP II to be bound in a well defined geometry, indicating the presence of specific protein-protein interactions at the docking site. At moderately reducing conditions, the bound HiPIP II donates electrons to the cytochrome subunit bound to the reaction center with a half-time of < or =11 micros. This donation reaction was analyzed by using Marcus's outer-sphere electron-transfer theory and compared with those observed in other HiPIP-containing purple bacteria. The results indicate substantial differences between the HiPIP- and the cytochrome c2-mediated re-reduction of the reaction center.

Fig. 1.

EPR spectra recorded on illuminated whole cells (trace a) and on chemically oxidized purified HiPIP I (trace b) and HiPIP II (trace c) from H. halophila. Instrument settings: microwave frequency, 9.44 GHz; temperature, 15 K; microwave power, 6.3 mW; modulation 1 mT.

Fig. 2.

EPR spectra recorded on membrane fragments and on increasingly enriched fractions of the membrane-bound HiPIP. Membrane fragments (spectrum a) were oxidized by addition of 2 mM ferricyanide in 50 mM Mops (pH 7), sedimented by ultracentrifugation, and resuspended in 50 mM Mops (pH 7). Spectrum b was recorded on a supernatant fraction obtained after sonication of these membranes. Spectrum c was recorded on the HiPIP fraction collected after DEAE-column chromatography of the supernatant fraction described above. Spectrum d was recorded on HiPIP collected from the size exclusion column at the end of the purification. Instrument settings were as in Fig. 1.

Fig. 3.

Redox titrations of HiPIP II in membrane fragments and in the soluble form. EPR titration on membrane fragments (open squares) was performed in the dark in 50 mM Mops, 130 g/liter NaCl (pH 7). The amplitude of the g = 2.10 peak measured on EPR spectra recorded as in Fig. 1 is plotted versus the ambient redox potential. For this particular experiment, the data points are fitted by an n = 1 Nernst curve with E_m =+125 mV. Optical redox titrations of purified HiPIP II were performed in 50 mM Mops (pH 7) in the absence (closed squares) or the presence (open circles) of 2.5 M NaCl. The A_{475 nm} - A_{600 nm} difference is plotted versus the ambient redox potential. The best fit in the absence of NaCl is obtained with E_m =+50 mV (n = 1). In the presence of NaCl, two equivalent components with n = 1 and E_m = +50 mV and +160 mV were required.

Fig. 4.

EPR spectra recorded on partially ordered membrane fragments from H. halophila. Spectra obtained on the chemically oxidized membranes show HiPIP signals with significant anisotropy as illustrated by selected spectra (trace a at 90° and trace d at 30°). Spectrum b obtained on ascorbate-reduced samples shows largely diminished HiPIP signals. The anisotropic signal reappears after illumination at 4°C of these ascorbate reduced membranes (spectra c and e). The additional lines at 315 mT and at 325 mT are due to manganese. (Inset) Polar plot evaluation of the dependence of the g₁ signal amplitude at 2.10 versus angle of the magnetic field with respect to the membrane plane. Filled squares represent data points of the chemically oxidized sample, and open squares represent data points of the photooxidized sample. Instrument settings are as in Fig. 1.

Fig. 5.

Kinetics of laser flash-induced absorption changes recorded on membrane fragments in 50 mM Mops, 130 g/liter NaCl (pH 7) at 607 nm (A) and at 422 nm (B). The ambient redox potential was poised to +100 mV (filled squares), +10 mV (filled triangles), or -40 mV (open circles). Kinetics were fitted by using four components with t_1/2 = 210 ns, 2.1 μs, 11–18 μs, and 11 μs. The relative amplitudes of the four phases vary as a function of ambient redox potential. The dotted simulation of the μs-phase at high redox potentials was obtained assuming an amplitude ratio between 607 nm and 422 nm kinetics equal to that of the fast (ns-) phase.