Cytochrome cbb3 oxidase and bacterial microaerobic metabolism

Tetrapyrroles: Their Life, Birth and Death

amine adduct, and did not lead to to the formation of Fe(I1)-NiR [lo]. So far, attempts to reduce crystalline Fe( I I I)-NiR to Fe( I I)-NiR, by reaction either with dithionite or with dihydrogen in the presence of traces of [Ni,Fe] dihydrogenase from S. deleyianum, have been unsuccessful. Usu- ally, the crystals begin to crack. Currently, soaking experiments are under way with Fe(II1)-NiR and several of the 0-substituted hydroxylamine de- rivatives, in an attempt to obtain a closer look at the binding of hydroxylamine to the active haem centre of NiR.

This work was supported by the Deutsche Forschungs- gerneinschaft, the German-Israel Foundation, and the Volkswagen Stiftung.

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Pettigrew, G. W. and Moore, G. R. ( 1987) Cytochrornes c: Biological Aspects, Springer-Verlag, Berlin, Heidelberg. New York, London, Paris, Tokyo Blackrnore, R. S., Gadsby, P. M. A,, Greenwood, C. and Thornson, A. J. ( 1990) FEBS Lett. 264, 257-262 Brittain, T., Blackmore, R. S., Greenwood, C. and Thornson, A. J. ( I 992) Eur. J. Biochern. 209, 793-802

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Received I I March 2002

Cytochrome cbb, oxidase and bacterial microaerobic metabolism R. S. Pitcher*', T. Brittaint and N. 1. Watmough"

"Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K., and +School of Biological Sciences, University of Auckland, Private Bag 920 19,

Auckland, New Zealand

Abstract central to its proposed role in bacterial micro- Cytochrome ebb, oxidase is a member of the aerobic metabolism. Recent spectroscopic charac- haem-copper oxidase superfamily. is charac- terization of both the cytochrome cbb, oxidase terized by its high oxygen while retaining complex from Pseudomonas stutzeri and the di- the ability to pump protons. These attributes are haem ccOp subunit expressed in

Escherichia coli has revealed the presence of a low- spin His/His co-ordinated c-type cytochrome. The low midpoint reduction potential of this haem Key words: cytochrorne oxidase, Pseudomonos, respiration, spec-

troscopy. Abbreviations used: HCO, haern-copper oxidase; SUI, subunit I. 'To whom COrresDondence should be addressed (e-mail

(Em < + 100 mV), together with its unexpected ability to bind c0 in the reduced state at the

r.pitcher(a) uea.ac uk). expense of the distal histidine ligand, raises ques-

653 0 2002 Biochemical Society

Biochemical Society Transactions (2002) Volume 30, part 4

tions about the role of the ccoP subunit in the delivery of electrons to the active site.

Introduction The final step in the electron transport chain of mitochondria and aerobically respiring bacteria is the four-electron reduction of dioxygen to water. This exogonic reaction is usually catalysed by members of the haem-copper oxidase (HCO) superfamily, integral membrane proteins that use the liberated free energy to translocate protons across the periplasmic membrane. In spite of differences in their immediate electron donors, affinity for oxygen and cofactor composition, all members of the HCO superfamily contain a highly conserved catalytic subunit (subunit I ; SUI), and are believed to be monophyletic in origin [l-31. SUI comprises at least 12 transmembrane helices and contains the active site, a bimetallic centre formed by the iron of a high-spin, penta-co- ordinated haem to which substrate and other exogenous ligands can bind, and an adjacent copper ion (Cu,) [4]. Also found in SUI is the binding site for a second haem, that is six-co- ordinate, which serves to transfer electrons to the active site. Transmembrane helices 11, VI, VII and X of SUI harbour six absolutely conserved histidine residues that are diagnostic of the entire superfamily and which serve to ligate both haem irons and Cu, [S].

The eubacterial HCOs can essentially be divided into two main groups: the quinol oxidases, e.g. Escherichia coli cytochrome bo,, and the cytochrome c oxidases, such as the cytochromes aa, from Paracoccus denitrificans and Rhodobacter sphaeroides. The main difference between the two types of oxidase is the presence of a fourth metal centre, known as Cu, or purple copper, in the hydrophilic domain of subunit I1 of cytochrome c

oxidases. Cu, contains two copper atoms in a mixed valence configuration [6,7] and is the primary acceptor of electrons from cytochrome c [8]. The Cu, centre is also found in bacterial nitrous oxide reductases [9], where it also serves as an electron receiver that is distinct from the catalytic centre, Cu, [lo]. Thus far, Cu, centres have not been described in HCOs that use quinols as the immediate electron donor, with the single exception of a menaquinol : NO oxidoreductase isolated from Bacillus azotoformans [ l 13.

In recent years a third highly divergent group of HCOs, the cytochrome cbb, oxidases, have been described in the proteobacteria [12]. Cytochrome cbb, oxidases have been purified from several

0 2002 Biochemical Society 654

species of proteobacteria, including Paracoccus denitrificans [ 1 31, Rhodobacter sphaeroides [ 141, Rhodobacter capsulatus [ 1 51, Bradyrhizobium japonicum [ 161 and Pseudomonas stutzeri [ 171. Rather poor yields and a tendency for the complex to dissociate has made detailed biochemical charac- terization difficult, and a structure is not yet available, although crystallization conditions have been reported [17]. However, there is evidence that the cytochrome cbb, oxidases are characterized by an unusually high affinity for oxygen ( K , - 7 nM) [16], combined with an ability to pump protons [ 181.

Analysis of proton translocation both in cell suspensions [13,18] and in purified cytochrome cbb, oxidase reconstituted into phospholipid ves- icles [19,20] suggests that the H+/e- stoichiometry of cytochrome cbb, is a variable number, usually between 0.2 and 0.5, which is significantly lower than the maximum efficiency ( 1 H+/e-) that is exhibited by cytochrome aag. Physiological studies are consistent with the notion that cytochrome cbb, is less efficient at transducing energy than cytochrome aa, [21], although a value of 1 H+/e- has been reported for the cytochrome cbb, oxidase from R . sphaeroides [22].

A specialized enzyme at the heart of microaerobic metabolism? Genes encoding a cytochrome cbb, oxidase were initially identified in B. japonicum [23] and desig- nated j i x N O Q P (ccoNOQP), since their expression is required both to support symbiotic N, fixation, which is energetically demanding, while ensuring that the 0,-labile nitrogenase is not compromised. More recently, homologous genes have been identified in other proteobacteria, e.g. R . capsulatus [24], Azorhizobium caulinodans [25] and a number of human pathogens, where it is presumed to play a role in the colonization of anoxic tissues. These include Campylobacter jejuni [26], Helicobacter pylori [27], Pseudomonas aeru- ginosa [28] and Vibrio cholerae [29]. The ccoNOQP operon encodes four non-identical subunits and is always found close to a second gene cluster known as ccoGHIS, the expression of which is necessary for the assembly of a functional cbb, oxidase [30,31].

The exclusive presence of cytochrome cbb, oxidase in the proteobacteria [2,12] is in stark contrast with the universal distribution of the HCO catalytic centre throughout all domains of life. This suggests that, far from being an evol-


utionary remnant related to the primordial oxidase that is proposed to have evolved some 2 billion years ago [3], cytochrome cbb, oxidase represents a modern enzyme that has evolved independently to fulfil a specialized role in microaerobic energy metabolism [2]. Moreover, cytochrome cbb:, oxidase is also a close relative of the equally specialist bacterial nitric oxide reductases, with which it shares phylogenetic origins [1,32] as well as a number of structural features [2,33]. In fact, a cytochrome cbb, oxidase has the highest N O reductase activity of any known true dioxygen- reducing HCO [34].

Identification of the six canonical histidine residues that ligate the metal centres of SUI led to a proposed topological model of ccoN based upon a minimum of 12 transmembrane helices [35,36]. These helices correspond to the 12 authentic helices seen in the X-ray structures of classical HCOs [37,38]. However, despite similar organization and conserved metal ligands, ccoN contains neither haem 0 nor haem A in the active site. The dinuclear centre consists of haem B (haem b,) that is magnetically coupled to Cu,. This is interesting, as haem B, unlike haem 0 and haem A, does not contain a hydroxyethylfarnesyl substituent of the porphyrin macrocycle, which has been implicated in proton movements [39]. Like E. colicytochrome bo,, the magnetically isolated low-spin haem of ccoN, haem b, is also a protohaem.

Another significant difference between ccoN and the catalytic subunit of other eubacterial oxidases is suggested by careful inspection of their primary sequences. Many of the ionizable residues that form two structurally defined proton uptake channels of cytochrome aa, oxidase, known as the D- and K-channels, which are responsible for moving protons from the face of the inner membrane to the buried dinuclear centre during turn- over, are absent from the derived amino acid sequence of ccoN [13]. In particular, tyrosine-280 (residue numbering corresponds to the P. denitri- $cans cytochrome aa, sequence), that lies at the top of the K-channel, is absent. In cytochrome aa,, post-translational modification of tyrosine- 280 covalently links it to histidine-284, one of the Cu, ligands [40], to form a site capable of stabi- lizing a radical species during oxygen reduction [39]. There is no obvious replacement for this unusual structure in the cytochrome cbb,-type oxidases, which might suggest that the design of the proton pump is fundamentally different, perhaps accounting for the different H'/e- stoichio- metries discussed above.

655

Organization of the cytochrome cbb, oxidase complex

Although cytochrome cbb, oxidases appear to utilize cytochrome c and not quinol [15,41] as an electron donor, they lack a Cu, site [14]. Instead the complex contains two membrane-anchored subunits that contain c-type haems. These are the monohaem cco0 (23 kDa) and the dihaem ccoP (35 kDa), one or both of which may serve to transfer electrons derived from the bc, complex to the catalytic subunit ccoN. Despite the presence of a single haem c binding site, cco0 exhibits minimal similarity to known c-type cytochromes [42], with the notable exception of norC, a membrane-anchored cytochrome c subunit of N O reductase [32]. Equilibrium redox titrations [15] identified two electrochemically distinct c-type cytochromes with reduction potentials (En,) of + 265 mV and + 320 mV and a reduction potential of +385 mV for haem b. Further titrations of a membrane fraction derived from a mutant (M7) that incorporates only cco0 into the enzyme complex allowed the assignment of the + 320 mV reduction potential to the c-type haem in this subunit [15]. Mutagenesis studies on the assembly and function of the individual subunits are also consistent with these assignments [43]. These results led to the proposal that electrons are passed from the electron donor to haem b via ccoP and cco0 in that order, although the requirement for such a degenerate electron transfer chain is far from clear.

The cytochrome cbb, oxidase operon includes a fourth gene, ccoQ, which is predicted to encode a small membrane-bound polypeptide. The presence of ccoQ in a purified complex has so far been demonstrated immunologically only in B. japonicum [36], and its function remains unclear. In- frame deletion mutants of ccoQ constructed in B. japonicum [43] and R. sphaeroides [44] have no apparent effects upon the assembly or the activity of cytochrome cbb, oxidase, although quantity of the cytochrome c appeared to be somewhat de- creased in the ccoQ mutant of B. japonicum [43]. There is some recent evidence to suggest that in R. sphaeroides ccoQ serves as a ' transponder ' in an as yet undefined signal transduction pathway that controls the expression of photosynthesis-related genes in response to the flux of electrons through the cytochrome cbb, oxidase. It has been suggested that this specific role for ccoQ may be related to the presence of two histidine residues that are conserved in R. sphaeriodes and R. capsulatas, but

0 2002 Biochemical Society


which are not present in non-photosynthetic species [44,45].

Cytochrome cbb, oxidase from Pseudornonas stutzeri Pseudomonas stutzeri is a facultative anaerobic bacterium that is capable of denitrification. Re- cently a preparation has been described that provides high yields of stable cytochrome cbb, oxidase from this organism [17]. We have recently prepared the enzyme using this method and assessed its integrity by a combination of SDS/ PAGE, oxygen uptake measurements, haem de- termination and M S [46]. In addition, we have sequenced the ccoNOQP operon of P. stutzeri, and the derived amino acid sequences are consistent with the experimentally determined subunit masses and haem determinations.

Detailed spectroscopic analysis of the oxidized cytochrome cbb, oxidase from P. stutzeri using a combination of UV/visible, EPR and room-temperature magnetic CD spectroscopies is consistent with the presence of four low- spin haems : haem b together with the three c-type haems of cc00 and ccoP. This analysis clearly showed that one of the c-type haems of either cco0 or ccoP had His/His co-ordination, rather than the rather more usual His/Met ligation. Room- temperature magnetic CD spectroscopy of ccoP that had been heterologously expressed in E. coli revealed that the origin of the His/His-co- ordinated c-type haem was this subunit. Cyto- chromes c that have His/His rather than His/Met ligation generally have a lower reduction potential (Em < 100 mV), due to a reduced ability to stabil- ize Fe(I1) [42]. This discovery was rather unexpected, because of the midpoint reduction potential of the presumed electron donor cytochrome c (Em approx. +250 mV), and poses the question of the likely role of the His/His-co- ordinated c-type haem in ccoP.

Past difficulties in obtaining cytochrome cbb, oxidase in high yield has made the spectroscopic study of the enzyme from other sources rather difficult. For example, there are only two published EPR spectra of the purified oxidized enzyme ; unfortunately, these are only of sufficient resolution to demonstrate the presence of low-spin ferric haem and confirm the absence of Cu, [14,15]. In fact, signals associated with each of the four low-spin ferric haems can clearly be seen in the X-band EPR spectrum of the P. stutzeri enzyme at 10 K, although unambiguous assign-

ments could only be made by reference to the EPR spectrum of ccoP.

Unusual CO binding properties of cytochrome cbb, oxidase We have also been able to use isolated ccoP to try and resolve some of the outstanding issues con- cerning the binding of CO to cytochrome cbb, oxidases. In our hands we found evidence of CO binding to both b- and c-type cytochromes (Fig- ure 1). This has also been observed in cytochrome cbb, oxidase from B. japonicum [30,41], while the published spectra of the enzyme purified from R. cupsulatus [15] indicate that CO bound only to a c-type cytochrome. When we examined the separately expressed ccoP subunit, we found that it can bind CO stoichiometrically, but only after reduction with dithionite. The fact that CO does not bind after reduction with ascorbate suggests that the low-potential bis-histidine-ligated c-type haem must be reduced in order to bind CO (Fig- ure 1). The separately expressed periplasmic soluble domain of ccoP from B. japonicum expressed in E. coli can also bind CO [47]. However, there is no evidence of CO binding to a c-type haem in the enzyme purified from P. denitri$cans, although this may be accounted for by close inspection of the reduced-minus-oxidized difference spectra [13,18], which show the ccoP subunit to be lost during purification [13,43].

Taking into account this new information, we have gone on to study the recombination of CO with both reduced ccoP and the active-site haem b,. The rate of CO recombination with the active- site haem b, (& = 7 x lo5 M-' . s-l ), as determined by laser-flash photolysis, is appreciably higher than for other HCOs that contain copper in their active site (kon = 7 x lo4 M-' . s-') ([48] and references therein), which is consistent with a somewhat more open dinuclear centre that allows CO to bind to ferrous haem b, in a linear, rather than a bent, conformation [49]. In contrast, the kinetics of CO recombination with ccoP are broadly similar to those observed in a recently characterized six-co-ordinate plant haemoglobin [SO]. This suggests that, in ccoP, CO binding must be preceded by displacement of the distal ligand from the His/His-co-ordinated haem. These ob- servations lead us consider whether ccoP serves not only as an electron transfer domain, but also as a sensory domain in a signal transduction pathway, perhaps involving ccoQ. Whatever the final answer, there is some way to go in elucidating the

0 2002 Biochemical Society 656


Figure I

Binding of CO to reduced haem centres in cytochrome cbb, oxidase from Pseudomonas stutzeri

In the isolated enzyme complex, CO can bind both to ferrous haem b, in the reduced dinuclear centre and to the His/His-ligated c-type haem in ccoP, giving nse to troughs in the reduced-CO minus reduced difference spectrum at 560 nm and 550 nm respectively (A) In isolated ccoP. CO binds only to the ferrous His/His-ligated c-type haem, giving nse to a single feature at 550 nm (8)

Cytochrome c& OxMase U

CCOP

functions(s) of this fascinating class of HCOs in microbial respiration.

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The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella

C. Schwalb*, S. K. Chapman? and G. A. Reid"' "Institute of Cell and Molecular Biology, University of Edinburgh, The Kings Buildings, Mayfield Road,

Edinburgh EH9 3JR, Scotland, U.K., and tDepartment of Chemistry, University of Edinburgh, The Kings Buildings, West Mains Road, Edinburgh EH9 311, Scotland, U.K.

Abstract Shewanella spp. demonstrate great variability in the use of terminal electron acceptors in anaerobic respiration ; these include nitrate, fumarate, DMSO, trimethylamine oxide, sulphur com-

Key words: anaerobic respiration, electron transport, flavocytochrome c,, haern, truncated CymA. Abbreviations used: 'CymA, truncated CyrnA (constructed by deleting the sequence encoding the first 35 amino acids of CymA) ; Fcc,, flavocytochrome cj. ITo whom correspondence should be addressed (e-mail [email protected]).

0 2002 Biochemical Society

pounds and metal oxides. These pathways open up possible applications in bioremediation. The wide variety of respiratory substrates for Shewan- ella is correlated with the evolution of several multi-haem membrane-bound, periplasmic and outer-membrane c-type cytochromes. The 21 kDa c-type cytochrome CymA of the freshwater strain Shewanella oneidensis MR-1 has an N-terminal membrane anchor and a globular tetrahaem periplasmic domain. According to sequence align- ments, CymA is a member of the NapC/NirT family. This family of redox proteins is responsible for electron transfer from the quinone

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