(29), and the modifications that distinguish phycocyanobilin from biliverdin are also at vinyl positions, we contemplate a formyl analog of biliverdin
as a plausible product candidate (fig. S7). Thus,
TSPO might catalyze both the vinyl-to-formyl oxidation and also the oxidative cleavage of PpIX
at the methene bridge between vinyl-bearing
A mechanistic clue for TSPO-mediated degradation of PpIX comes from the observation that
the W→F mutation of a conserved tryptophan
was said to abrogate PpIX degradation by Ct TSPO
without affecting PpIX binding (12). In our Bc TSPO-PpIX model (fig. S6C), both the corresponding
residue, Trp138, and another conserved tryptophan,
Trp51, are poised for potential interactions with
the scissile methene bridge and with the vinyl
groups. To examine the possible roles of these
residues in PpIX degradation, we compared the
reaction for the wild type with reactions for the
W138F, W51F, W51F/W138F variants, and we also
tested A142T because of its relation to the polymorphic variation at the analogous position (A/T147)
in Homo sapiens TSPO1 (Hs TSPO1). We used the
sensitive PpIX fluorescence spectrum (27, 28) to
monitor the changes.
The prominent fluorescence features of free
PpIX were shifted slightly and quenched appre-
ciably (~30%) upon interaction with wild-type
(WT) BcTSPO, and these features disappeared
within minutes during light exposure (Fig. 3, C
and D). Preincubation with saturated PK11195
slowed the decay of PpIX greatly (Fig. 3D) but
did not block it entirely. As for Ct TSPO, the W138F
mutant of Bc TSPO greatly reduced the catalytic
degradation of PpIX compared with the WT pro-
tein (Fig. 3, E and F); however, in this case, the
degradation was not abolished altogether. For
the W51F mutant, we saw no evidence of the
product formed by WT and W138F Bc TSPO. In-
stead, we observed a rich fluorescence spectrum
(Fig. 3, G and H) with new peaks growing at 649
and 673 nm as the primary peak at 632 nm
decreased. We attribute the new peaks to sec-
ondary excited states, probably due to incipient
photooxidation (29). These states are intrinsic to
PpIX (fig. S8, A and B) but stabilized by TSPO
(Fig. 3G and fig. S8C) and reversed on PpIX dis-
sociation (fig. S8, D and E). We conclude that
Trp51 is essential for PpIX cleavage and that
Trp138 participates in the reaction.
Neither the W51F/W138F double mutant nor
the A142T showed any PpIX cleavage, but each
generated reversible excitation at 673 nm as the
primary 632-nm fluorescence decreased (Table
1 and fig. S9, A to D). To test for the greater generality of TSPO-mediated PpIX reactions, we produced various eukaryotic TSPO homologs. Among
these we have been able to extract and purify three
vertebrate proteins: Xenopus tropicalis TSPO
(Xt TSPO), Hs TSPO1 A147T, and Hs TSPO2. From
the sequences (fig. S1) considered in light of our
analysis of Bc TSPO properties, we expected that
Xt TSPO should degrade PpIX similarly to WT
Bc TSPO but that the other two would show aberrant behavior. Indeed, Xt TSPO degraded PpIX,
as monitored by our fluorescence assay, whereas
both HsTSPO1 (Thr147) and Hs TSPO2 (Thr145)
showed growth of 673-nm fluorescence at the
expense of 632-nm decay but no irreversible degradation (Table 1 and fig. S9, E to J). Because the
latter behavior is as for Bc TSPO A142T, we infer
that having threonine at position 142 precludes
cleavage-appropriate binding of PpIX in each of
them. Protein instability complicated our analysis of reaction rates for the vertebrate TSPOs.
What is the chemical basis for TSPO-mediated
PpIX degradation? The intrinsic photochemistry
of PpIX involves the generation of singlet oxygen
1O2 and other reactive oxygen species (ROS) upon
photostimulation (27–30), and it is evident from
our observations that excited states of PpIX are
stabilized when bound to TSPOs. Additionally,
ROS interactions with tryptophan residues can
generate tryptophan radicals, which are essential
for the oxidation of aromatic substrates by lignin
peroxidases (31). We suggest that such tryptophan radicals at Trp51 and Trp138 (Bc TSPO numbering) may be responsible for TSPO-mediated
oxidation of PpIX. Radical interactions may work
at a distance, and conserved Trp31 and Trp40 may
also play a role (fig. S10). Although ROS come from
light in our in vitro experiments, ROS-generating
cellular processes may be responsible in vivo.
What physiological role might PpIX degrada-
tion serve? Porphyrins such as PpIX are tightly
controlled because they can generate toxic ROS,
especially when excited by light. An incisive anal-
ysis of the role of TSPO in such control came in
studies of oxidative stress in the moss Physcomi-
trella patens (32). Mitochondrial Pp TSPO1, which
has catalytic residues the same as in Bc TSPO (fig.
S1), was shown to be induced by oxidative stress
from the inhibition of mitochondrial respiration,
and knockout lines showed an accumulation of
PpIX and ROS. In other studies, porphyrins ex-
erted toxic effects on liver in the dark, generating
hydroxyl radicals (33). Biliverdin is an oxyradical
scavenger and is cytoprotective (34). We suggest
that TSPO-mediated degradation of PpIX to
bilindigin both reduces ROS generation and also
promotes ROS neutralization.
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554 30 JANUARY 2015 • VOL 347 ISSUE 6221 sciencemag.org SCIENCE
Table 1. Reactions of PpIX with TSPO proteins. Down arrows denote the decrease with time in
intensity of photostimulated fluorescence from the first excited state. Up arrows denote the increase
with time of fluorescence from secondary excited states, presumably reached by oxidative
632 nm 649 nm 673 nm
Bc TSPO wild type Fast ↓ No No
Bc TSPO wild type No ↓ ↑ ↑
Bc TSPO wild type Slow ↓ No No
Bc TSPO W51F/W138F No ↓ No ↑
Bc TSPO A142T No ↓ No ↑
XtTSPO Yes ↓ No No
Hs TSPO1 T147 No ↓ No ↑
Hs TSPO2 T145 No ↓ No ↑