Novel FixL homologues in Chlamydomonas reinhardtii bind heme and O2
U.M. Narayana Murthya, Matt S.A. Weckerb, Matthew C. Posewitzc, Marie-Alda Gilles-Gonzalezd,
Maria L. Ghirardia,⇑
aBiosciences Center, National Renewable Energy Laboratory, Golden, CO 80401, United States
bGeneBiologics LLC, Boulder, CO 80303, United States
cDepartment of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO 80401, United States
dDepartment of Biochemistry, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, TX 75390-9038, United States
a r t i c l ei n f o
Received 1 May 2012
Revised 26 June 2012
Accepted 28 June 2012
Available online 16 July 2012
Edited by Stuart Ferguson
a b s t r a c t
Genome inspection revealed nine putative heme-binding, FixL-homologous proteins in Chlamydo-
monas reinhardtii. The heme-binding domains from two of these proteins, FXL1 and FXL5 were
cloned, expressed in Escherichia coli, purified and characterized. The recombinant FXL1 and FXL5
domains stained positively for heme, while mutations in the putative ligand-binding histidine
FXL1-H200S and FXL5-H200S resulted in loss of heme binding. The FXL1 and FXL5 [Fe(II), bound
O2] had Soret absorption maxima around 415 nm, and weaker absorptions at longer wavelengths,
in concurrence with the literature. Ligand-binding measurements showed that FXL1 and FXL5 bind
O2with moderate affinity, 135 and 222 lM, respectively. This suggests that Chlamydomonas may use
the FXL proteins in O2-sensing mechanisms analogous to that reported in nitrogen-fixing bacteria to
regulate gene expression.
? ? 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Chlamydomonas is able to perform photosynthesis and aerobic
respiration, transition into strictly anaerobic fermentations when
O2is unavailable, and balance fermentation, photo-fermentation
and respiration under conditions of sulfur deprivation in the light
[1–3]. The anaerobic metabolism of phototrophic microorganisms
has been of particular interest for the production of organic acids,
alcohols and H2, all of which can be used in strategies for the pro-
duction of renewable fuels [4–8]. Several studies have defined as-
pects of these metabolic capabilities in Chlamydomonas; however,
relatively little is known about the mechanisms of metabolite sens-
ing or the signal-transduction events that occur in response to O2
levels. Recent data indicate that significant changes occur in the
abundance of several transcripts encoding fermentative enzymes
as Chlamydomonas acclimates to anoxia [6,9]. Therefore, we ana-
lyzed the available Chlamydomonas genome for homologues of
known O2-sensing proteins and signal-transduction components
that have been characterized in other organisms. We identified a
group of Chlamydomonas genes that are predicted to encode pro-
teins with strong amino acid similarity to the Rhizobial heme-
binding, O2-sensing PAS domains. The expected proximal histidine
residue (H200 in BjFixL) is present in all of the Chlamydomonas
FXL homologues, as are two highly conserved arginines (R206
and R220 in BjFixL) known to be involved in hydrogen-bonding
interactions with the heme. From this set of Chlamydomonas
FixL-like (FXL) homologues, we chose two members, FXL1 and
FXL5 for further studies regarding their potential role as O2sensors
and gene-expression regulators in Chlamydomonas.
The full-length versions of FXL1 and FXL5 proteins in Chla-
mydomonas are very large (2072 and 2299 amino acids, respec-
tively) and each of the putative homologues has multiple
transmembrane-spanning domains, which are typical of the bacte-
rial FixL homologues. To better understand the role of heme pro-
identified PAS domains were able to bind O2, the putative heme-
binding domains from FXL1 and FXL5 proteins were cloned, heter-
ologously expressed in Escherichia coli, and purified. The purified
FXL1 and FXL5 proteins were then characterized for their heme
and O2-binding properties. Our results clearly indicate that FXL1
and FXL5 bind heme and, like their Rhizobial homologues, could
be involved in heme-based O2-sensing and the regulation of asso-
ciated metabolic pathways in Chlamydomonas. However, since the
Chlamydomonas FXL homologues lack canonical autophosphoryla-
tion and signal transmitter domains, they must utilize an unusual
signal transduction mechanism involving additional residues/
0014-5793/$36.00 ? 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
⇑Corresponding author. Address: National Renewable Energy Laboratory, 1617
Cole Blvd, Golden, CO 80401, United States. Fax: +1 303 384 7836.
E-mail addresses: firstname.lastname@example.org, email@example.com (M.L. Ghir-
FEBS Letters 586 (2012) 4282–4288
journal homepage: www.FEBSLetters.org
2. Materials and methods
2.1. Sequence analysis, alignments and protein modeling
Chlamydomonas FXL sequence analysis was performed using
the DNASTAR expert sequence analysis software (Madison, WI).
Searches for sequence similarity were performed using the BLAST
network service provided by the National Center for Biotechnology
Information (www.ncbi.nlm.nih.gov). Sequence alignment was
performed using the MegAlign tool of DNASTAR. Chlamydomonas
FXL proteins models were analyzed using the Chlamydomonas
v4.3 genome portalathttp://www.phytozome.net/chlamy.
Sequence for the FXL6 homologue was obtained using the
Chlamydomonas version 3.0 gene model since the current model
of FXL6 excludes a stretch of nucleotide sequence at scaf-
fold_48:736979–737039 by instead assigning these nucleotides
to an intron that shows high sequence similarity to the other rep-
resented FXL proteins. Regions of identity in protein sequence were
assessed by alignment using EMBL-EBI clustalW v2 (http://www.
ebi.ac.uk/Tools/msa/clustalw2/) and overall alignment identity
percentages were assessed with Kalign (http://msa.cgb.ki.se/cgi-
bin/msa.cgi) using a gap open penalty of 11, a gap extension pen-
alty of 0.85, a terminal gap penalty of 0.45 and a bonus score of 0
2.2. Plasmid construction
The primer sequences used in this work are listed in Supple-
mentary Table 1. RNA was extracted from Chlamydomonas strain
CC425 and reverse-transcribed as described . For expression in
E. coli, the heme-binding domain of FXL1 was amplified using
primers FXL1BamR and FXL1NdeF. Similarly, the heme binding do-
main of FXL5 was amplified using primers FXL5BamR and
FXL5NdeF. The corresponding PCR products were digested with
BamHI and NdeI and then ligated into pET28a (Novagen, Madison,
WI) for expression as His6-tagged proteins. FXL1 and FXL5 mutant
constructs were also generated in which the putative heme-bind-
ing histidine was changed to serine, creating FXL1-H200S and
FXL5-H200S. To change the single histidine to serine in the FXL do-
main of FXL1 and FXL5 recombinant PCR was performed using the
appropriate primer sets shown in Supplementary Table 1 in two
consecutive reactions that generated the mutated domains and
then annealed and further amplified them with the appropriate
T7 primers. The resulting PCR products were digested with BamI
and NdeI and then ligated into pET28a (Novagen, Madison, WI)
for expression as His6-tagged proteins.
2.3. Protein purification
For purification of the FXL1 and FXL5 heme-binding domains,
the plasmids containing the FXL1 or FXL5 sequences were trans-
formed into E. coli BL21 (DE3) codon+(Stratagene, La Jolla, CA)
and expression was induced by IPTG. The FXL1 and FXL5 domains
were purified by the procedure developed by Murthy et al. .
The purified proteins were dialyzed against 25 mM Tris–HCl, pH
7.4. Homogeneity and purity assessments of all proteins employed
SDS–PAGE with Coomassie blue staining. In addition, LC–MS/MS
(done by the Colorado State University at Fort Collins, CO according
to the method described in http://www.pmf.colostate.edu/) and
UV–visible spectroscopy were carried out after the final elution.
2.4. Gel heme assays
Proteins were analyzed using 15% Tris–Tricine mini gels with a
6% stacking gel in Tris–Tricine buffer containing 0.05% SDS. Protein
bands were visualized after staining for heme with o-dianisidine
(DMB) according to the procedure developed by Francis and Becker
2.5. Spectrophotometric studies and estimation of O2dissociation
Absorption spectra (350–700 nm) of samples at 25 ?C in 50 mM
phosphate buffer (pH 7.4) were recorded with a Varian 4000 spec-
trophotometer. The deoxygenated [Fe(II), no bound O2] spectra
were recorded after reduction with a twofold molar excess of so-
dium dithionite followed by removal of the reductant through a
3 mL G-25 column equilibrated with degassed buffer at 4 ?C. To ob-
tain the oxy-FXL [Fe(II), bound O2] absorption spectra while avoid-
ing auto-oxidation, one atmosphere of O2was layered over the
deoxygenated samples and the solutions were equilibrated by
shaking immediately before the spectra were recorded. Oxidized
spectra [Fe(III)] for FXL1 and FXL5 were obtained by exposing the
samples to air for 30 min or treating them with potassium ferricy-
anide at 25 ?C. Apoproteins were prepared by extracting heme
from FXL1 and FXL5 by cold acid-acetone treatment as described
previously . The determination of the stoichiometric amount
of heme bound to each protein was done according to the method
of Atassi and Childress .
In order to estimate O2dissociation constants, a stock solution
of 1.3 mM O2in 50 mM phosphate buffer (pH 7.4) was prepared
by bubbling with O2at room temperature for 1 h in a septum-
sealed glass vial. The stock solution was then transferred to an
anaerobic chamber, and various aliquots were transferred to deox-
ygenated buffer samples in individual sealed glass vials using a
gas-tight Hamilton syringe to prepare an O2dilution series. Puri-
fied FXL1 and FXL5 protein samples in the deoxy state were added
to respective vials of the dilution series and their absorption spec-
tra recorded. Spectra for deoxy and oxy states of the protein using
titrations from 0 to 1280 lM O2were used to determine O2satura-
tion , and O2equilibrium dissociation constants (Kdvalues).
2.6. Quantitative real-time PCR
RNA was extracted from Chlamydomonas reinhardtii strain
CC425 as described  and DNaseI was used to remove DNA before
reverse transcription. Real-time PCR (RT-PCR) was performed using
a LightCycler 480 in combination with the SYBRGreen I Master kit
(Roche, Germany) according to the supplier’s protocol.
3.1. Identification of FXL homologues in Chlamydomonas and sequence
Nine FXL-like sequences were identified by BLAST searches of
the Chlamydomonas genome data base JGI v4.3 using Rhizobium
FixL as a query. Interestingly, these genes aligned closely with at
least five hypothetical genes (VOLCADRAFT-104960, -104036,
-93382, -91579 and -93917) from Volvox carteri, a colonial chloro-
phyte alga, closely related to the single-celled Chlamydomonas.
These potential Volvox FXL genes retained the canonical heme-
binding histidine of the Chlamydomonas FXL proteins (not shown).
As with the Chlamydomonas FXL genes, these potential Volvox
genes did not contain the FixL histidine phosphorylation site.
Each Chlamydomonas protein contained a FixL-like heme-bind-
ing PAS consensus site (Figs. 1B and 2). The nine FXL protein PAS
core regions, comprising about 100 residues between the two ends
of the predicted b-sheet structures of the PAS domain shared 76%
overall amino acid identity. These same regions showed a 59%
U.M.N. Murthy et al./FEBS Letters 586 (2012) 4282–4288
overall identity when three Rhizobial FixL heme-binding PAS do-
mains were included in the alignment. Nearly all of the main fea-
tures of the FixL heme-binding sites are present (see Fig. 1
legend). Importantly, the Bradyrhizobium japonicum BjFixL H200
PAS residue is conserved, as is a putative axial binding Histidine li-
gand to the Fe atom. As mentioned above, all nine homologues
lacked canonical phosphorylation and DNA-binding domains,
although it is worth noting that the FXL6 C-terminal domain has
high sequence similarity to ankyrin protein–protein interaction
domain (Supplementary Fig. 1), and a potential Ser phosphoryla-
Fig. 1. Structural elements implicated in BjFixL regulation and their occurrence in other heme-PAS proteins. (A) Contrast of the crystal structure of the wild-type BjFixLH ‘‘on-
state’’ (left) and ‘‘off-state’’ (right). This figure is adopted with permission from Gilles-Gonzalez et al. . (B) Amino acid sequence similarity of the nine FXL Chlamydomonas
proteins to the PAS domains of selected Rhizobia FixL. The position numbering at the end of each line refers to the amino acid position number for the modeled protein.
Arrows above the notations refer to the a-helix turns and b-sheet structures. Number notations below the sequences refer to the following conserved heme-PAS residues (1)
residues lying in the plane of the porphyrin ring. (2) Residues that are peripheral to heme coordination. (3) Residues that orient BjH200 to the proximal side of the heme
group. (4) Strictly conserved histidine that is required for coordination to heme. (5) Arginine residue that binds the edge of the heme and coordinates ligand detection with
the FG loop transmitter. (6) Residues that bind propionate group on heme. (7) Arginine that binds the heme propionate in the ‘‘on-state’’ or O2in ‘‘off-state’’ and transmits its
status to the FG loop in Bj. (8) Residues oriented on the distal side of the heme. (9) Phosphorylation site. The sequences for the FXL1PAS and FXL5PAS truncates begin at the 1st
amino acid shown and extend to the end of the I b-sheet structure.
U.M.N. Murthy et al./FEBS Letters 586 (2012) 4282–4288
tion residue is observed downstream from the PAS consensus do-
main in FXL3. Schematic representation of the deduced sequence
of the nine C. reinhardtii FIXL homologues and the locations of
the predicted functional elements are indicated in Fig. 2.
3.2. Characteristics of heterologously-expressed FXL heme-binding
The purified FXL1 and FXL5 were orange–red proteins with
masses of about 12.5 kDa as judged by SDS–PAGE (Supplementary
Fig. 2A). The purified FXL1-H200S and FXL5-H200S proteins, with
substitutions of the proximal histidine to serine were colorless,
indicating the absence of heme. LC–MS/MS analysis of FXL1 and
FXL5 indicated that the respective proteins were correctly purified
(Supplementary Fig. 3A and B). His6tag-cleaved FXL1 and FXL5
were used for all biochemical characterizations. Purified FXL1
and FXL5 carried heme throughout the purification step. Purified
FXL1 and FXL5, as well as FXL1H-200S and FXL5-H200S were as-
sayed for heme-binding by using a non-denaturing gel heme assay
(Supplementary Fig. 2B). As expected, FXL1 and FXL5 both stained
positively for heme, whereas FXL1-H200S and FXL5-H200S showed
no heme reaction.
Hemin titrations show that FXL1 and FXL5 interact with hemin
in a 1:1 M ratio of hemin to protein (Supplementary Fig. 2C and D).
The concentration of the FXL1 and FXL5 protein calculated from
the absorbance at 415 nm agree with the protein concentration
determined from the protein assay, suggesting that FXL1 and
FXL5 bind one heme per monomer.
3.3. Spectral properties and ligand binding properties
Absorption spectra were collected in the range of 350–700 nm
before and after reduction of FXL1 and FXL5 proteins from the fer-
ric form [Fe(III)] to the ferrous form [Fe(II)]. The optical absorption
of met [Fe(III)], deoxy [Fe(II), no bound O2] and oxy [Fe(II), bound
O2], spectra complexes of FXL1 and FXL5 are shown in Fig. 3A
and B. The Soret and visible absorption peaks of oxy (415 nm)
and deoxy (430 nm) Fe(II) complexes of FXL1 and FXL5 are charac-
teristic of protein-bound heme and are similar to those of deoxy
and oxy RmFixL . Upon reduction with 10 mM sodium dithio-
nite the primary 415 nm Soret bands of both FXL1 and FXL5 shift
about 15 nm downfield, and the ratio of the absorbance of the
560 nm band relative to the 530 nm band increases.
Linear combinations of spectra for deoxy and oxy states of the
protein were used to determine the saturations at varying O2con-
centrations (Fig. 3C and D). FXL1 and FXL5 showed hyperbolic O2-
saturation curves (Fig. 3E and F). From this study, we determined
the equilibrium Kd for O2 binding to FXL1 to be ?135 lM. For
FXL5, the Kdvalues were determined to be ?222 lM.
The presence of nine FXL homologues in Chlamydomonas sug-
gests that this alga may use heme-based O2sensing to regulate as-
pects of metabolism in response to O2. Particularly intriguing is a
possible role in sensing and responding to a transition from an aer-
obic to an anaerobic metabolism.
250 5007501000 12501500 17502000 22502500
Upstream consensus site
Fig. 2. Schematic representation of the nine C. reinhardtii FXL homologues, as predicted by Chlamydomonas V4.3 website. Only FXL1 and FXL5 have been accurately
sequenced from cDNA. Approximate locations of the functional elements described in Fig. 1 are indicated.
U.M.N. Murthy et al./FEBS Letters 586 (2012) 4282–4288
4.1. Heme-binding proteins in Chlamydomonas: sequence analysis
Despite retaining many features central to the function of the
bacterial FixL proteins, several unique features are observed in
the Chlamydomonas homologues that may be a consequence of
expression and function in an eukaryotic host. The histidine re-
quired for autophosphorylation and signal transduction in bacte-
rial FixL is absent from the Chlamydomonas FXL homologues.
Therefore it is not clear how the putative Chlamydomonas FXL
PAS domains might relay the state of the heme ligand to a regula-
tory element. Chlamydomonas FXL proteins are the only known
members of the heme family lacking histidine kinase domains for
autophosphorylation (although the five hypothetic genes from Vol-
vox that show homology to FXL sequences also lack the canonical
His phosphorylation residue). We predict that other functional do-
mains exist in these FXL proteins, which might relay the state of
the heme ligand. For example, bacterial PAS elements lacking his-
tidine kinase domains are often associated with phosphodiesterase
or DNA-binding domains . We speculate on the possibility that
signal transduction functions are performed by other residues/do-
mains present in the Chlamydomonas FXL homologues, such as the
ankyrin domain present in the model of FXL6 (Supplementary
Fig. 1A). Ankyrin domains have been shown to bind to a number
of plasma membrane-associated proteins , including the Na/
K-ATPase , the amiloride-sensitive sodium channel  and
the voltage-dependent sodium channel . Recently, it has been
Fig. 3. Spectral properties and ligand binding properties of the FXL homologues. Characteristic UV–visible absorption spectra of FXL1 (A) and FXL5 (B) proteins. Blue line,
deoxy [(Fe(II), no bound O2]; red line, oxy [Fe(II), bound O2]; green line, met [Fe(III)]. Changes in FXL1 (C) and FXL5 (D) absorption spectra upon titration with O2. Absorption
spectra of FXL1 and FXL5 are shown at various O2-saturation levels (inlM). For FXL1, the spectrum under 320 lm O2is not included. However, the absorption values from the
second replicate were used to calculate the Kdvalues. For FXL5, only one replicate was used at the 20 lM O2concentration. All other spectra contain at least two replicates for
FXL1 and FXL5. Details are in Section 2. Determination of the FXL1 (E) and FXL5 (F) O2affinities. Kdvalues of 135 and 222 lM (average of two replicates) for FXL1 and FXL5,
respectively were determined by directly titrating both proteins with 0–1280 lM O2, deconvoluting the deoxy and oxy fractions in the absorption spectra, and fitting the data
to a hyperbolic equation for single binding, using least-square analysis.
U.M.N. Murthy et al./FEBS Letters 586 (2012) 4282–4288
shown that ankyrin also interacts with intracellular calcium chan-
nels such as the IP3receptor . 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 O2. 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
The Kdvalues for O2binding to FXL1 and FXL5 were 135 and
222 lM, respectively. The estimated Kdvalue for FXL1 is close to
that found in the Bradyrhizobium FixL (140 lm) but much higher
than in Rhizobium FixL (0.003 lm) [15,24]. The Kdvalue for FXL5
is much higher than the values calculated for Bradyrhizobium
and Rhizobium but much lower than E. coli DOSH (340 lm)
[15,25,26] (see Supplementary Table 2). It is also important to note
that the Kdvalues, 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 O2levels and initiates the appropri-
ate transcriptional, translational and posttranslational responses.
The Kdvalues for both FXL domains are near the concentration of
O2dissolved in water in equilibrium with the atmosphere. The rel-
atively high Kdvalues for O2suggest that the FXL proteins are used
to respond to changing levels of O2at the soil surface or to O2pro-
duced during photosynthesis. Since Chlamydomonas can generate
significant quantities of photosynthetic O2, the expression of O2-
sensitive proteins must be tightly controlled to ensure that cellular
energy is not wasted on the synthesis of O2-intolerant proteins
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
by O2and their transcription is down-regulated by O2[27,28]. Sim-
ilar challenges are faced by N2-fixing Rhizobia, some of which use
the heme-based, O2-sensing FixL proteins to detect O2levels and
initiate signal transduction events that ensure the synthesis of N2
fixation proteins only when O2levels 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 O2levels.
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.
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