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 (Kd=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 [13]. 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 structure

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 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/).

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 membrane-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 (Kd=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 (Kd) 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 Kd 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 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 NADPH-dependent nitrate reductase. In this way, the reduction of nitrate to nitrite is accompanied by a decrease in NADPH concentration, which was confirmed spectrophotometrically.

Kinetic analyses of SHP

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.

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.

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.

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.

A possible mechanism for NO dioxygenation as catalysed by the SHP/DHC system

Figure 3
A possible mechanism for NO dioxygenation as catalysed by the SHP/DHC system

The numbering scheme is fully described in the text.

Figure 3
A possible mechanism for NO dioxygenation as catalysed by the SHP/DHC system

The numbering scheme is fully described in the text.

Why is a nitric oxide dioxygenase necessary?

Although NO is an important signalling molecule, it is also a ubiquitous poison. At concentrations lower than 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 [1113]. 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 physiological relevance of these findings. To this end, several knockout mutants are being studied to determine their phenotypes.

Integration of Structures, Spectroscopies and Mechanisms: Second Joint German/British Bioenergetics Conference, a Biochemical Society Focused Meeting held at University of Edinburgh, U.K., 2–4 April 2008. Organized by Ulrich Brandt (Frankfurt, Germany), Steve Chapman (Edinburgh, U.K.), Peter Heathcoate (Queen Mary, University of London, U.K.), John Ingledew (St Andrews, U.K.), Mike Jones (Bristol, U.K.), Bernd Ludwig (Frankfurt, Germany), Fraser MacMillan (University of East Anglia, Norwich, U.K.), Hartmut Michel (Max-Planck-Institute for Biophysics, Frankfurt am Main, Germany), Peter Rich (University College London, U.K.) and John Walker (MRC Dunn Human Nutrition Unit, Cambridge, U.K.). Edited by Ulrich Brandt and Peter Rich.

Abbreviations

     
  • 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

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