Resolution and Reconstitution of a Bound Fe−S Protein from the Photosynthetic Reaction Center ofHeliobacterium modesticaldum

Abstract

The photosynthetic reaction center of Heliobacterium modesticaldum (HbRC) was isolated from membranes using n-dodecyl beta-D-maltopyranoside followed by sucrose density ultracentrifugation. The low-temperature EPR spectra of whole cells, isolated membranes, and HbRC complexes are similar, showing a single Fe-S cluster with g values of 2.067, 1.933, and 1.890 after illumination at 20 K, and a complex spectrum attributed to exchange interaction from two Fe-S clusters after illumination during freezing. The protein containing the Fe-S clusters was removed from the HbRC by washing it with 1.0 M NaCl and purified by ultrafiltration over a 30 kDa cutoff membrane. Analysis of the filtrate by SDS-PAGE showed a major band at approximately 8 kDa that was weakly stained with Coomassie Brilliant Blue and strongly stained with silver. The optical spectrum of the oxidized Fe-S protein shows a maximum at 410 nm, and the EPR spectrum of the reduced Fe-S protein shows a complex set of resonances similar to those found in 2[4Fe-4S] ferredoxins. The HbRC core was purified by DEAE ion-exchange chromatography and resolved by SDS-PAGE. The purified HbRC was composed of a band at ca. 40 kDa, which is identified as PshA, and several additional proteins. The isolated Fe-S protein rebinds spontaneously to purified HbRC cores, and the light-induced EPR signals of the Fe-S clusters are recovered. The flash-induced kinetics of the HbRC complex show two kinetic phases at room temperature, one with a lifetime of 75 ms and the other with a lifetime of 15 ms. The 75 ms component is lost when the Fe-S protein is removed from the HbRC complex, and it is regained when the Fe-S protein is rebound to HbRC cores. Thus, the 75 ms kinetic phase is derived from recombination of a terminal Fe-S cluster with P798(+), and the 15 ms kinetic phase is derived from recombination with an earlier acceptor, probably F(X). We suggest that the bound Fe-S protein present in the HbRC be designated PshB.

Biogenesis of Iron-Sulfur Clusters in Photosystem I

“…ance changes in the near-IR were measured for reconstituted PS I complexes with a laboratory-built, double-beam spectrophotometer as described (6,31), except that the cuvettes used were 2 ϫ 10 mm instead of 5 ϫ 10 mm. The samples were prepared under anoxic conditions, with the following final concentrations: 1 M P700-F X cores, 20 M apo-PsaC, 20 M PsaD, 42 M holo-NfuA, 10 M DCPIP, 10 mM ascorbate, 4 mM DTT, and 0.04% (w/v) DM.…”

“…Low-temperature X-band EPR Spectroscopy of Holo-NfuAThe method for low-temperature, X-band EPR spectroscopy has been described (31). Measurements were performed using an ECS-106 X-band spectrometer (Bruker Biospin, Billerica, MA) equipped with an ESR900 liquid helium cryostat and an ITC-4 temperature controller (Oxford Instruments, Billerica, MA).…”

The bound iron–sulfur clusters of Type-I homodimeric reaction centers

“…NaCl concentrations as low as 100 mM were found to result in the loss of 50% of the EPR signal intensity of F A and F B . These results were explained by the dissociation of the protein that harbors the F A and F B clusters from the HbRC core (Heinnickel et al 2005). Because previous attempts to isolate the HbRC often used ionic detergents such as deriphat 160c (Trost and Blankenship 1989) as well as relatively high ionic strengths, it is not surprising that the protein containing the F A and F B clusters was not present after purification.…”

“…In 2005, Heinnickel et al (2005) published a method by which the F A /F B protein could be removed from the HbRC. The resulting HbRC core showed monophasic charge recombination kinetics with a 15-ms lifetime at room temperature, a value which is similar to that reported previously in urea treated membranes (Kleinherenbrink et al 1994).…”

Crystal structure of Tflp: A ferredoxin‐like metallo‐β‐lactamase superfamily protein from Thermoanaerobacter tengcongensis

“…The protocol for the reconstitution of [Fe-S] cluster has been described by Heinnickel et al 13 Tflp under dithionite-reduced condition was incubated with 1 mM FeCl 3 in 20 mM phosphate buffer (pH 7.0) for 20 min. Subsequently, Na 2 S was added to a final concentration of 1 mM.…”