An electrostatic surface analysis of the HbRC
reveals that the N-side surface is mostly positively
charged, with two positive patches close to FX
and an extended Lys on either side (Fig. 5). Homology models generated for PshB1 (see supplementary materials) predict a negatively charged
surface that may interact with these patches,
facilitating reduction by FX.
Heliobacteria use cytochrome c553 (cyt c553) as
an electron donor to the HbRC. This is similar in
structure to cyt c6 (56) but is attached to the
membrane through covalent linkage to a diacyl-glycerol (42, 43), thus restricting its diffusion to
two dimensions. The surface closest to P800 is
fairly neutral (Fig. 5). There are two surface
helices at the P-side of P800 that would serve as
the possible cyt-binding site. Although similar to
that of cyanobacterial PSI (12), the helix in the
HbRC is shorter and has mainly polar uncharged
side chains (Ser, Thr, Asn, Gln) pointing away
from P800, and the Trp that is important for cyt
binding in PSI (44, 45) is absent.
The lower surface charge of the HbRC relative to PSI may provide a rationale for asymmetry
in the oxygen-tolerant RCs. The HbRC appears to
interact with electron acceptors and donors through
hydrophobic and polar uncharged surfaces rather
than ionic interactions, which would require forming ion bridges between the symmetric surface
of the HbRC and the monomeric—and thus inherently asymmetric—electron-carrier proteins.
The evolution of asymmetry may have provided an
advantage in binding electron donors and acceptors more tightly, based on specific ionic interactions between complementary surfaces.
The positions occupied by the PshX subunits
are similar to the sites where PsaI (near PsaB)
and PsaJ (near PsaA) are found in PSI (Fig. 2A).
Both of these are single-TMH subunits, like
PshX. PshX and PsaJ both coordinate two
antenna BChls, but only one of these is in the
same location, and even in this case, the axial
ligand is not conserved in the sequence between
the HbRC and PSI. PsaI does not coordinate any
antenna chlorophylls. Further, there is very low
sequence identity shared between PshX and either
PsaI (9%) or PsaJ (10%). Thus, it seems likely that
the appearance of PshX is a case of convergent
evolution driven by a functional requirement. As
with PsaI and PsaJ, a single carotenoid is wedged
between the single-TMH subunit and the periphery of the core dimer (lime in Fig. 1A). Carotenoids
are found in PSI between the TMH of all transmembrane peripheral subunits and the TMH of
PsaA-PsaB, implying that, in addition to their
functional role, they play a structural role; and
this may be the case in the HbRC as well. However, there are only two carotenoids in the HbRC,
in contrast to the 22 carotenoids in PSI, and they
are shorter as well (30 instead of 40 carbons).
This likely reflects the environmental niche of
heliobacteria, which are strict anaerobes and
therefore do not need to prevent the formation
of potentially harmful singlet oxygen.
One might expect the HbRC to display charac-
teristics more similar to the common ancestor of
all RCs because of its overall simplicity: its homo-
dimeric core, the low number of antenna BChls
relative to its oxygenic homolog, and its lack of
peripheral antenna complexes or other subunits
(other than PshX) (46). The conjecture that a
homodimeric RC should have preceded a hetero-
dimeric RC in the evolutionary trajectory is also
consistent with this idea. However, the HbRC
has had a long time to evolve from the ancestral
RC and has certainly acquired unique features
that are advantageous to the organism. It has
been proposed that the Firmicutes, within which
the family Heliobacteriaceae resides, branched
early in bacterial evolution (7). Despite this, the
ancestor of heliobacteria probably did not origi-
nate the first RC but instead acquired it through
horizontal gene transfer, consistent with the
colocation of the pshA gene in one gene cluster
along with all the genes required for pigment
synthesis (47). However, some traits of the last
common ancestor of all RCs may be preserved
in the HbRC as a result of its host’s anoxic niche,
which has similarities with early Earth.
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We are grateful to J. Whitelegge for his validation of the PshX
sequence by mass spectrometry and to Y. Mazor for modeling and
graphical assistance. This work was funded by the Division of
Chemical Sciences, Geosciences, and Biosciences, Office of Basic
Energy Sciences, of the U.S. Department of Energy (DOE)
through grant DE-SC0010575 to K.E.R., R.F., and J.H.G. and
supported with x-ray crystallographic equipment and infrastructure
provided by P. Fromme of the Biodesign Center for Applied
Structural Discovery at Arizona State University. The Berkeley
Center for Structural Biology is supported in part by the National
Institutes of Health, National Institute of General Medical
Sciences, and the Howard Hughes Medical Institute. The Advanced
Light Source is a DOE Scientific User Facility supported by the
Director, Office of Science, Office of Basic Energy Sciences and
operated for the DOE Office of Science by Lawrence Berkeley
National Laboratory. Results shown in this report are derived from
work performed at Argonne National Laboratory, Structural
Biology Center at the Advanced Photon Source. The Structural
Biology Center is funded by the DOE Office of Science, Office of
Biological and Environmental Research. Argonne is operated by
UChicago Argonne, LLC, for the DOE Office of Science under
contract DE-AC02-06CH11357. K.E.R., J.H.G., and R.F. designed
the project. I.S. and C.G. performed purifications, their subsequent
optimization, and characterization. C.G., B.F., and R.F. optimized
crystallization conditions. C.G. crystallized HbRC that was used in
x-ray diffraction experiments. R.F. and C.G. collected x-ray
diffraction data. R.F. and C.G. built models for molecular
replacement phasing of diffraction data. R.F. and C.G. performed
model building and refinement. C.G., K.E.R., and R.F. wrote the
manuscript with input from all other authors. The HbRC structure
has been deposited into the Protein Data Bank with accession
code 5V8K. The authors declare no competing financial interests.
Materials and Methods
Figs. S1 to S8
Tables S1 to S4
27 May 2017; accepted 19 July 2017
Published online 27 July 2017