shown that ankyrin also interacts with intracellular calcium chan-
nels such as the IP
receptor . Indeed, ankyrin domains are
found in the Chlamydomonas copper responsive regulator 1
(CRR1) that is associated with anoxic response . Finally, the
100 amino acid consensus sequence closely upstream of the
PAS domain in each of the FXL proteins (see Supplementary
Fig. 1B), does not share strong identity to any known protein. Fur-
ther work is needed to identify the elements of the full signal
transduction pathway involving FXL proteins in Chlamydomonas.
4.2. FXL1 and FXL5 are heme-binding proteins
Recent microarray data indicated that only slight increases oc-
cur in the abundance of two of the nine FXL proteins, namely
FXL1 and FXL5, as Chlamydomonas acclimates to anoxia . In this
study, using highly puriﬁed proteins, we showed that their expres-
sion level remains unchanged upon anaerobiosis (not shown) and
that they are heme-binding domains (Supplementary Fig. 2B). A
change of histidine to serine eliminates heme-binding for FXL1-
H200S and FXL5-H200S mutants. This position is thus likely to
be the site of heme–iron coordination and provides further evi-
dence for the importance of this histidine residue in heme coordi-
nation. Finally, we show that FXL1 and FXL5 each bind one mole of
hemin per mole of protein to form a stable hemin-protein complex
(Supplementary Fig. 2C and D).
4.3. Spectral properties of the FXL1 and FXL5 heme-binding domains
are typical of FixL proteins
Spectral measurements of the recombinant FXL1 and FXL5
heme-binding domains show intense absorption at around
415 nm (the ‘‘Soret’’ band), followed by weaker absorptions at
longer wavelengths. These are characteristic bands of protein-
bound heme in the oxy Fe[II] form and are very similar to absorp-
tion spectra of Rhizobium FixL protein  both in the presence and
absence of O
. This was conﬁrmed by the 15 nm downﬁeld shift
seen upon reduction (deoxy [FeII] form), which is also typical of
heme-binding proteins. Heme proteins with cysteines, methionine
or tyrosine as proximal ligands, on the other hand, have very differ-
ent absorption spectra . Our absorption data and the sequence
homology with Rhizobium FixL suggest that the heme moieties in
FXL1 and FXL5 are present in a binding environment similar to that
of the FixL heme.
4.4. Possible physiological roles of FXL proteins in Chlamydomonas
values for O
binding to FXL1 and FXL5 were 135 and
M, respectively. The estimated K
value for FXL1 is close to
that found in the Bradyrhizobium FixL (140
m) but much higher
than in Rhizobium FixL (0.003
m) [15,24]. The K
value for FXL5
is much higher than the values calculated for Bradyrhizobium
and Rhizobium but much lower than E. coli DOSH (340
[15,25,26] (see Supplementary Table 2). It is also important to note
that the K
values, while serving well to indicate the saturation
state of the hemes, do not necessarily relate linearly to the activi-
ties of the proteins. In the case of RmFixL, pronounced hysteretic
behavior was reported, such that relatively low saturation of the
heme could completely shut down the kinase activity. Based on se-
quence similarity to the Rhizobium FixL protein, and from the re-
sults presented above, it is reasonable to assume that the two
proteins described here function as heme-binding proteins in vivo.
Given the metabolic ﬂexibility of Chlamydomonas and its ability to
transition quickly from an aerobic to an anaerobic environment
(and vice versa), it will be critical to understand the mechanisms
by which the organism senses O
levels and initiates the appropri-
ate transcriptional, translational and posttranslational responses.
values for both FXL domains are near the concentration of
dissolved in water in equilibrium with the atmosphere. The rel-
atively high K
values for O
suggest that the FXL proteins are used
to respond to changing levels of O
at the soil surface or to O
duced during photosynthesis. Since Chlamydomonas can generate
signiﬁcant quantities of photosynthetic O
, the expression of O
sensitive proteins must be tightly controlled to ensure that cellular
energy is not wasted on the synthesis of O
during aerobic growth. Similarly, it is essential for aerobically
growing Chlamydomonas to down-regulate the expression of pro-
teins that are required to be functional only during anaerobiosis.
For example, the [FeFe]-hydrogenases are irreversibly inhibited
and their transcription is down-regulated by O
ilar challenges are faced by N
-ﬁxing Rhizobia, some of which use
the heme-based, O
-sensing FixL proteins to detect O
initiate signal transduction events that ensure the synthesis of N
ﬁxation proteins only when O
levels are sufﬁciently low to pre-
vent enzyme inactivation. It is thus tempting to propose that the
Chlamydomonas homologues are involved in regulating transcrip-
tion of genes in response to changes in intracellular O
This work was supported by the a grant from the Chemical
Sciences, Geosciences and Biological Sciences Program, Division
of Energy Biosciences, Ofﬁce of Science, U.S. Department of Energy.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.febslet.2012.
 Antal, T.K., Krendeleva, T.E., Laurinavichene, T.V., Makarova, V.V., Ghirardi,
M.L., Rubin, A.B., Tsygankov, A.A. and Seibert, M. (2003) The dependence of
algal H2 production on Photosystem II and O2 consumption activities in
sulfur-deprived Chlamydomonas reinhardtii cells. Biochim. Biophys. Acta 1607,
 Fouchard, S., Hemschemeier, A., Caruana, A., Pruvost, J., Legrand, J., Happe, T.,
Peltier, G. and Cournac, L. (2005) Autotrophic and mixotrophic hydrogen
photoproduction in sulfur-deprived chlamydomonas cells. Appl. Environ.
Microbiol. 71, 6199–6205.
 Melis, A., Zhang, L., Forestier, M., Ghirardi, M.L. and Seibert, M. (2000)
Sustained photobiological hydrogen gas production upon reversible
inactivation of oxygen evolution in the green alga Chlamydomonas
reinhardtii. Plant Physiol. 122, 127–136.
 Gfeller, R.P. and Gibbs, M. (1984) Fermentative metabolism of Chlamydomonas
reinhardtii: I. Analysis of fermentative products from starch in dark and light.
Plant Physiol. 75, 212–218.
 Kruse, O., Rupprecht, J., Mussgnug, J.H., Dismukes, G.C. and Hankamer, B.
(2005) Photosynthesis: a blueprint for solar energy capture and biohydrogen
production technologies. Photochem. Photobiol. Sci. 4, 957–970.
 Mus, F., Dubini, A., Seibert, M., Posewitz, M.C. and Grossman, A.R. (2007)
Anaerobic acclimation in Chlamydomonas reinhardtii: anoxic gene expression,
hydrogenase induction and metabolic pathways. J. Biol. Chem. 282, 25475–
 Posewitz, M.C., King, P.W., Smolinski, S.L., Zhang, L., Seibert, M. and Ghirardi,
M.L. (2004) Discovery of two novel radical S-adenosylmethionine proteins
required for the assembly of an active [Fe] hydrogenase. J. Biol. Chem. 279,
 Posewitz, M.C., Smolinski, S.L., Kanakagiri, S., Melis, A., Seibert, M. and
Ghirardi, M.L. (2004) Hydrogen photoproduction is attenuated by disruption
of an isoamylase gene in Chlamydomonas reinhardtii. Plant Cell 16, 2151–
 Dubini, A., Mus, F., Seibert, M., Grossman, A.R. and Posewitz, M.C. (2009)
Flexibility in anaerobic metabolism as revealed in a mutant of Chlamydomonas
reinhardtii lacking hydrogenase activity. J. Biol. Chem. 284, 7201–7213.
 Lassmann, T. and Sonnhammer, E. (2005) Kalign – an accurate and fast
multiple sequence alignment algorithm. BMC Bioinformatics 6, 298.
 Murthy, U.M.N. et al. (2009) Characterization of Arabidopsis thaliana SufE2 and
SufE3. Functions in chloroplast iron-sulfur cluster assembly and NAD
synthesis. J. Biol. Chem. 284, 27020.
U.M.N. Murthy et al. / FEBS Letters 586 (2012) 4282–4288