992
Biochemical Society Transactions (2008) Volume 36, part 5
Rhodobacter sphaeroides haem protein: a novel
cytochrome with nitric oxide dioxygenase activity
Bor-Ran Li*, J.L. Ross Anderson*, Christopher G. Mowat*, Caroline S. Miles†, Graeme A. Reid†
and Stephen K. Chapman*1
*EaStCHEM, School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, U.K., and †Institute of Structural and Molecular Biology,
University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, U.K.
Abstract
Rhodobacter sphaeroides produces a novel cytochrome, designated as SHP (sphaeroides haem protein),
that is unusual in having asparagine as a redox-labile haem ligand. The gene encoding SHP is contained
within an operon that also encodes a DHC (dihaem cytochrome c) and a membrane-associated cytochrome
b. DHC and SHP have been shown to have high affinity for each other at low ionic strength (K d = 0.2 μM),
and DHC is able to reduce SHP very rapidly. The reduced form of the protein, SHP2+ (reduced or ferrous SHP),
has high affinity for both oxygen and nitric oxide (NO). It has been shown that the oxyferrous form,
SHP2+ –O2 (oxygen-bound form of SHP), reacts rapidly with NO to produce nitrate, whereas SHP2+ –NO
(the NO-bound form of SHP) will react with superoxide with the same product formed. It is therefore
possible that SHP functions physiologically as a nitric oxide dioxygenase, protecting the organism against
NO poisoning, and we propose a possible mechanism for this process.
Why is SHP so interesting?
SHP (sphaeroides haem protein; from the photosynthetic bacterium Rhodobacter sphaeroides) belongs to a family of O2 binding c-type cytochromes [1–3]. As yet, no physiological
role has been determined for the protein, but structural, kinetic and genetic information provides clues as to its function.
SHP has an unprecedented structure
The crystal structure of SHP has been solved in the oxidized
[SHP3+ (oxidized or ferric SHP)] and reduced [SHP2+
(reduced or ferrous SHP)] forms [2,4], and was found to be
structurally similar to class I cytochromes c. In the oxidized
(SHP3+ ) form, the haem iron is six-co-ordinate, with Asn88
acting as a haem ligand (Figure 1). However, the structure of
SHP2+ reveals that Asn88 no longer binds to the haem, thus
leaving the iron available to bind diatomic species (Figure 1).
Indeed, the structures of reduced SHP in complex with nitric
oxide (NO) or cyanide are also available [4].
SHP is the only c-type cytochrome that utilizes asparagine
as a haem ligand, but the conservation of this ligand among
SHPs from 16 different species [2], and its unusual lability,
raise questions concerning its functional relevance.
SHP appears to be part of an electron
transfer pathway
The gene encoding SHP is part of an operon that includes
genes encoding DHC (dihaem cytochrome c) and a memKey words: crystal structure, c-type cytochrome, dihaem cytochrome c, nitric oxide dioxygenase,
Rhodobacter sphaeroides, sphaeroides haem protein.
Abbreviations used: cyt b, cytochrome b; DHC, dihaem cytochrome c; SHP, sphaeroides haem
protein; SHP2+ , reduced or ferrous SHP; SHP2+ –NO, the nitric oxide-bound form of SHP; SHP2+ –O2 ,
the oxygen-bound form of SHP; SHP3+ , oxidized or ferric SHP; SOD, superoxide dismutase.
1
To whom correspondence should be addressed (email [email protected]).
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Authors Journal compilation brane-bound cyt b (cytochrome b) [5]. The structure of
DHC has been solved to 1.85 Å resolution (1 Å = 0.1 nm),
and the interaction between SHP and DHC has been
characterized [5]. It has been demonstrated that SHP and
DHC have great affinity for each other at low ionic strength
(K d = 0.20 ± 0.01 μM; in 10 mM Hepes buffer, pH 7.2,
at 25◦ C). Furthermore, DHC can transfer electrons to
SHP very rapidly, with a second-order rate constant of
1.8 · 107 M−1 · s−1 (pH 7.2, 10◦ C, I = 0.5 M). The reduction
potentials of DHC and SHP are also suitably ordered for a
favourable electron transfer reaction, with the DHC haems
having reduction potentials of −310 and −240 mV, and SHP
having a potential of −105 mV. These potentials remain
unaltered upon complex formation. This evidence leads to
the possibility that SHP, DHC and cyt b may be functionally
linked, with cyt b perhaps acting as the reductant of DHC,
which in turn reduces SHP.
What is the function of SHP?
The ability of SHP2+ to bind diatomic molecules raises the
question of whether SHP possesses enzymatic activity
towards such species.
SHP binds O2 and NO with high affinity
The dissociation constant (K d ) for O2 binding to SHP2+ is
in the region of 20 μM [5]. Like other ferrous haem proteins,
SHP2+ has even greater affinity for NO than O2 , with the K d
for NO binding being sub-micromolar in magnitude [5]. In
fact, the SHP2+ –NO (the nitric oxide-bound form of SHP)
produced may be considered as a dead-end complex under
these conditions, similar to observations made for other
O2 -binding cytochromes [6]. The high affinity of SHP2+ for
Biochem. Soc. Trans. (2008) 36, 992–995; doi:10.1042/BST0360992
Integration of Structures, Spectroscopies and Mechanisms
Figure 1 SHP structure
(A) The overall structure of SHP (PDB ID 1DW0; [4]). (B) The haem
in the oxidized protein (PDB ID 1DW0; [4]). Asn88 can be seen to
ligate the haem iron via the side-chain amide oxygen. (C) The reduced
haem. The asparagine no longer ligates the haem iron, leaving it
available for binding of small molecules (PDB ID 1DW3; [4]). This Figure
was generated using PyMOL (DeLano Scientific; http://pymol.
sourceforge.net/).
Figure 2 Kinetic analyses of SHP
(A) A typical time-course trace for the reaction of SHP2+ –O2 with NO.
Decrease in the absorbance at 540 nm was used for monitoring decay
of SHP2+ –O2 . (B) The black trace represents an equimolar mixture of
SHP2+ –NO and SHP2+ –O2 . With time, this converts into the spectrum
of SHP3+ (grey trace), and nitrate is produced. (C) The best-fit trace
with closed circles represents the decay of SHP2+ –NO in the SHP2+ –NO/
SHP2+ –O2 mixture in the absence of SOD and catalase. The best-fit trace
with open circles represents the same process in the presence of SOD
and catalase, and shows no trace of SHP2+ –NO decay.
both of these diatomic molecules leads to the possibility that
SHP may display some catalytic activity towards either (or
both) of them.
SHP catalyses the reaction of NO with O2
to form nitrate
Recent stopped-flow experiments have demonstrated that
SHP2+ –O2 (the oxygen-bound form of SHP) reacts with
NO (Figure 2A) with observed rates in the millisecond
region (at 10◦ C). The formation of nitrate was confirmed
by incubation of the reaction mixture (after SHP had been
removed by centrifugation) with NADPH and NADPHdependent nitrate reductase. In this way, the reduction of
nitrate to nitrite is accompanied by a decrease in NADPH
concentration, which was confirmed spectrophotometrically.
Interestingly, however, mixing SHP2+ –NO with O2 does
not lead to any reaction, even with O2 in vast excess. This
is similar to observations made for flavohaemoglobin Fe2+ –
NO species [7].
SHP2+ –NO will react with superoxide
to form nitrate
This lack of reactivity of SHP2+ –NO towards O2 is in line
with the results from previous work on NO dioxygenation
by haem proteins [8]. However, when SHP2+ –NO is
exposed to superoxide, nitrate is again produced. Further to
this, it has been observed that on mixing SHP2+ –NO and
SHP2+ –O2 , catalysis occurs. In this case, SHP2+ –O2 acts as
a source of superoxide, decaying to SHP3+ . The superoxide
evolved then reacts with SHP2+ –NO to form nitrate as
before. Figure 2(B) shows the initial spectrum obtained on
mixing an equal amount of SHP2+ –O2 and SHP2+ –NO
(black trace), while the grey trace shows the final composition of the reaction mixture. This corresponds to the
formation of SHP3+ . As a control, the same experiment was
carried out in the presence of SOD (superoxide dismutase)
and catalase (Figure 2C). In this case, no decay of SHP2+ –NO
is observed, thus confirming that the reaction is mediated by
superoxide release from SHP2+ –O2 .
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Biochemical Society Transactions (2008) Volume 36, part 5
Figure 3 A possible mechanism for NO dioxygenation as catalysed by the SHP/DHC system
The numbering scheme is fully described in the text.
What is the mechanism of SHP-catalysed
NO dioxygenation?
The mechanism of NO dioxygenation by haem proteins is
generally believed to involve the Fe2+ –O2 complex reacting
with NO to produce nitrate and the oxidized cytochrome.
In the case of SHP, we propose a possible mechanism for
NO dioxygenation that involves the formation of a putative
SHP3+ –peroxynitrite intermediate (Figure 3). Our postulated
mechanism involves the reduction of SHP3+ [1] by some
donor (believed to be DHC in the physiological situation),
as mentioned in the subsection ‘SHP appears to be part of an
electron transfer pathway’ to form SHP2+ [2]. Once SHP is
reduced, Asn88 is remote from the haem iron, and SHP2+ can
bind O2 to form SHP2+ –O2 [3], which would autoconvert
into the superoxy-ferric complex, SHP3+ –O2 − [4]. This form
can then react with NO to give the ferric peroxynitrite species
[5], which will release nitrate and regenerate SHP3+ [1]. In
addition to this pathway, we propose a second route that
involves binding of NO to SHP2+ to form the SHP2+ –NO
complex [6]. This ferrous NO-bound species can then react
with superoxide produced by the decay of SHP3+ –O2 − [4]
to form a ferrous peroxynitrite intermediate [7], which can
subsequently release nitrate and rejoin the cycle.
Why is a nitric oxide dioxygenase
necessary?
Although NO is an important signalling molecule, it is
also a ubiquitous poison. At concentrations lower than
C The
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Authors Journal compilation 100 nM, NO is known to inactivate [4Fe–4S]-containing
hydratases. NO will also compete with dioxygen binding to
metalloproteins such as haemoglobin and myoglobin. For
these reasons, some organisms require an NO-metabolizing
enzyme, such as nitric oxide dioxygenase and/or nitric oxide
reductase, to defend against NO poisoning [9,10].
Conclusion
The mechanism described in the section ‘What is the mechanism of SHP-catalysed NO dioxygenation’ is likely to be similar to the one that operates in other nitric oxide dioxygenases
such as flavohaemoglobin, which is found in unicellular prokaryotic and eukaryotic organisms [11–13]. Previous work
has also shown that neuroglobin has nitric oxide dioxygenase
activity [14]. Like flavohaemoglobin, ferrous neuroglobin
also binds NO rapidly and irreversibly. The NO-bound
form is able to react with O2 , but does so slowly [14]. For this
reason the NO-bound form is considered a ‘frozen’ form,
without activity. The main function of neuroglobin is thought
to be in scavenging NO. Although no superoxide generation
has been reported for neuroglobin, it is structurally similar to
other cytochromes that are able to generate superoxide. It is
therefore conceivable that NO-bound neuroglobin may, like
SHP and possibly flavohaemoglobin, react with endogenous
superoxide via the mechanism proposed in Figure 3.
The discovery of nitric oxide dioxygenase activity in SHP
represents the initial characterization of this system, and
there is much work to be done to understand further the
Integration of Structures, Spectroscopies and Mechanisms
physiological relevance of these findings. To this end, several
knockout mutants are being studied to determine their
phenotypes.
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Received 5 June 2008
doi:10.1042/BST0360992
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