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: email@example.com, firstname.lastname@example.org (M.L. Ghir-
FEBS Letters 586 (2012) 4282–4288
journal homepage: www.FEBSLetters.org
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U.M.N. Murthy et al./FEBS Letters 586 (2012) 4282–4288