Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light-activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities.

Heliobacteria contain Type I reaction centers (RCs) and a homodimeric core, but unlike green sulfur bacteria, they do not contain an extended antenna system. Given their simplicity, the heliobacterial RC (HbRC) should be ideal for the study of a prototypical homodimeric RC. However, there exist enormous gaps in our knowledge, particularly with regard to the nature of the secondary and tertiary electron acceptors. To paraphrase S. Neerken and J. Amesz (2001 Biochim Biophys Acta 1507:278-290): with the sole exception of primary charge separation, little progress has been made in recent years on the HbRC, either with respect to the polypeptide composition, or the nature of the electron acceptor chain, or the kinetics of forward and backward electron transfer. This situation, however, has changed. First, the low molecular mass polypeptide that contains the terminal FA and FB iron-sulfur clusters has been identified. The change in the lifetime of the flash-induced kinetics from 75 ms to 15 ms on its removal shows that the former arises from the P798+ [FA/FB]- recombination, and the latter from P798+ FX- recombination. Second, FX has been identified in HbRC cores by EPR and Mössbauer spectroscopy, and shown to be a [4Fe-4S]1+,2+ cluster with a ground spin state of S=3/2. Since all of the iron in HbRC cores is in the FX cluster, a ratio of approximately 22 Bchl g/P798 could be calculated from chemical assays of non-heme iron and Bchl g. Third, the N-terminal amino acid sequence of the FA/FB-containing polypeptide led to the identification and cloning of its gene. The expressed protein can be rebound to isolated HbRC cores, thereby regaining both the 75 ms kinetic phase resulting from P798+ [FA/FB]- recombination and the light-induced EPR resonances of FA- and FB-. The gene was named 'pshB' and the protein 'PshB' in keeping with the accepted nomenclature for Type I RCs. This article reviews the current state of knowledge on the structure and function of the HbRC.

Type I homodimeric reaction centers, particularly the class present in heliobacteria, are not well understood. Even though the primary amino acid sequence of PshA in Heliobacillus mobilis has been shown to contain an F(X) binding site, a functional Fe-S cluster has not been detected by EPR spectroscopy. Recently, we reported that PshB, which contains F(A)- and F(B)-like Fe-S clusters, could be removed from the Heliobacterium modesticaldum reaction center (HbRC), resulting in 15 ms lifetime charge recombination between P798(+) and an unidentified electron acceptor [Heinnickel, M., Shen, G., Agalarov, R., and Golbeck, J. H. (2005) Biochemistry 44, 9950-9960]. We report here that when a HbRC core is incubated with sodium dithionite in the presence of light, the 15 ms charge recombination is replaced with a kinetic transient in the sub-microsecond time domain, consistent with the reduction of this electron acceptor. Concomitantly, a broad and intense EPR signal arises around g = 5 along with a minor set of resonances around g = 2 similar to the spectrum of the [4Fe-4S](+) cluster in the Fe protein of Azotobacter vinelandii nitrogenase, which exists in two conformations having S = (3)/(2) and S = (1)/(2) ground spin states. The Mössbauer spectrum in the as-isolated HbRC core shows that all of the Fe is present in the form of a [4Fe-4S](2+) cluster. After reduction with sodium dithionite in the presence of light, approximately 65% of the Fe appears in the form of a [4Fe-4S](+) cluster; the remainder is in the [4Fe-4S](2+) state. Analysis of the non-heme iron content of HbRC cores indicates an antenna size of 21.6 +/- 1.1 BChl g molecules/P798. The evidence indicates that the HbRC contains a [4Fe-4S] cluster identified as F(X) that is coordinated between the PshA homodimer; in contrast to F(X) in other type I reaction centers, this [4Fe-4S] cluster exhibits an S = (3)/(2) ground spin state.

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.