Pentahaem cytochrome c nitrite reductase

Tetrapyrroles: Their Life, Birth and Death

Pentahaem cytochrome c nitrite reductase: reaction with hydroxylamine, a potential reaction intermediate and substrate M. Rudolfr, 0. Einslet, F. Neesef and P. M. H. Kroneck*' *Fachbereich Biologie, Universitat Konstanz, Fach M665,D-78457 Konstanz, Germany, +Howard Hughes Medical Institute and Division of Chemistty and Chemical Engineering, California Institute of Technology, Pasadena, CA 9 I 125, U.S.A.,and f Max-Planck-lnstitut fur Strahlenchemie, Stiftstrasse 34-26, 45470 Mulheim an der Ruhr, Germany are involved in electron transfer and redox chemistry of inorganic nitrogen and sulphur compounds. These proteins contain conserved structural motifs of haem centres, despite significant differences in primary sequence and protein structure [8]. Another prominent representative within this class of multihaem enzymes is hydroxylamine oxidoreductase (HAO), which catalyses the oxidation of hydroxylamine to nitrite [9] :

Abstract T h e pentahaem enzyme cytochrome c nitrite reductase catalyses the reduction of nitrite to ammonia, a key reaction in the biological nitrogen cycle. T h e enzyme can also transform nitrogen monoxide and hydroxylamine, two potential bound reaction intermediates, into ammonia. Structural and mechanistic aspects of the multihaem enzyme are discussed in comparison with hydroxylamine oxidoreductase, a trimeric protein with eight haem molecules per subunit.

NH,OH

Based on X-ray crystallographic, spectroscopic and kinetic investigations of NiR, the reduction of nitrite co-ordinated to the Fe( I I)-Lys active site [2,10] has been suggested to begin with the heterolytic cleavage of the N-0 bond. T w o rapid one-electron reductions will lead to the production of an {FeNO}' species and, by protonation, to a H N O adduct. A further two-electron two-proton step will produce hydroxylamine, the only bound intermediate, which loses water to give the final product, ammonia [lo]. Interestingly, NiR can also reduce hydroxylamine and nitric oxide to ammonia, with specific activities of 50 "4 for hydroxylamine but only 1.5 'lofor nitric oxide relative to that for nitrite [ l l ] . T o get a deeper insight into the mechanism of action of NiR, also in comparison with that of HAO, and to learn more about the role of the bound reaction intermediate and substrate NH,OH [2,10], we began to investigate the interaction of oxidized [Fe( I I I)] and reduced [Fe( II)] NiR with hydroxylamine and derivatives. Note that, chemically, hydroxylamine usually acts as a strong reducing agent, but can also function as an oxidant, depending on the metal, co-ordinated ligands and external conditions such as pH. As oxidation products, N,, N,O, NO, NO,, NO, or NO; can be formed; the reduction product appears to be solely ammonia. In the case of Fe(II1) complexes, hydroxylamine will usually act as a strong reductant and the corresponding Fe( 11) compounds will be formed [12].

Introduction Dissimilatory nitrate-to-ammonia reduction represents an important branch of the biogeochemical nitrogen cycle. With nitrite as the only liberated intermediate [l], nitrate is first reduced to nitrite by a molybdenum-dependent nitrate reductase, and subsequently nitrite is converted into ammonia by cytochrome c nitrite reductase (NiR) : NO;

+ H,O + HNO, + 4e- + 4H'

+ 6e-+ 8H+ + NH: + 2H,O

-

Cytochrome c NiRs are pentahaem enzymes with interesting spectroscopic properties [2]. They have a molecular mass of x 5 5 kDa, are encoded by a single gene, nrfA [3], and have been found so far in proteobacteria belonging to subdivisions y (Escherichia coli [4,5] and Haemophilus infEuenzae Rd [6]) and E (Sulfurospirillum deleyianum [2] and Wolinella succinogenes [7]). NiR belongs to a growing family of structurally well characterized multihaem proteins that

Key words: dissimilatory nitrate + ammonia reduction, haemhaem interaction, hydroxylamine oxidoreductase, iron-lysine centre. Abbreviations used: NiR, nitrite reductase; HAO, hydroxylamine oxidoreductase. 'To whom correspondence should be addressed (e-mail peter.kroneck(a)uni-konstanzde).

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0 2002 Biochemical Society

Biochemical Society Transactions (2002) Volume 30, part 4

Experimental

[S]. These multihaem NiRs (NrfA) are homodimers, with ten haems in remarkably close packing, with Fe-Fe distances of between 9.3 and 12.8 A. T h e active-site haem shows an unusual lysine co-ordination, with a sp3-hybridized amine nitrogen as the proximal ligand (Figure 1). Substrates bind to the distal site of this haem, which is accessible from the protein surface through two narrow channels. T h e substrate inlet channel, with a positive electrostatic potential, prefers anions, whereas the product outlet channel, with a negative electrostatic potential, prefers cations. T h e active site can accommodate anions and uncharged molecules [8]. Crystal structures were obtained with nitrite, sulphite, sulphate, azide and hydroxylamine bound to the Fe( I I I)-Lys centre [lo]. T h e other four haems are bis-histidinyl coordinated, and are linked to the peptide backbone by thioether bonds to the cysteine residues of a classical haem-binding motif for periplasmic proteins, Cys-Xaa,-Xaa,-Cys-His. With edge-toedge distances of less than 4 direct 71-electron interactions between porphyrins become possible, and most likely functionally important. Close to the active haem centre a Ca2+-binding site was detected [2,5,8], which represents an essential structural feature in the overall architecture of the enzyme. Such a critical role for a Ca2+ ion in structural properties was recently discussed for horseradish peroxidase [ 161. HAO, which functions as an oxidase, is a trimer of polypeptides, with each monomer containing eight haems bound covalently via two cysteine thioether linkages, for a total of 24 haems per H A 0 trimer. Seven of these are c-type haems, while the eighth is the P460 haem centre [9,17]. Haem P460 possesses a covalent linkage between the 5-meso carbon of the porphyrin and the C-3 ring carbon of Tyr-467 (Figure 1). T h e fifth coordination position of P460 is occupied by a

Materials Growth of cells, and purification and crystallization of soluble NiR (NrfA) from S . deleyianum and W . succinogenes were carried out as described previously [2,8,13,14]. All chemicals were reagent grade or better.

UVlvisible spectra All measurements were carried out under the strict exclusion of dioxygen, in an atmosphere of NJH, (95 "&/5 o/o). Oxidized NiR (enzyme as isolated) was reduced photochemically in the presence of oxalate and 5-deazaflavin [15]. T h e protein was present at 5 p M in 1 0 0 m M potassium phosphate buffer, pH 7.0; substrate was freshly prepared (1 .O M in water) and added in 5 pl steps to give a final concentration of 50 m M . T h e spectra were recorded on a Cary 50 instrument equipped with a thermostatted cell holder (Varian, Darmstadt, Germany) at 25 "C, in 1.0 cm Thunberg cuvettes.

A,

Results and discussion Three-dimensionalstructures of NiR and H A 0 As expected from amino acid sequence comparisons [8], all of the distinguishing features observed in the three-dimensional structure of cytochrome c NiR [crystallized in the oxidized Fe(II1) state] from S . deleyianum are apparent in the structures of NiRs from W . succinogenes and E. coli. T h e residues that surround the active Fe( 111)-lysine haem centre, and form the substrate/product channel, are well conserved in the structures of the resolution) enzymes from S. deleyianum (1.9 [2], W . succinogenes (1.6 [8] and E . coli (2.5

A)

A

A)

Figure I Comparison of the active sites of NiR (left) and H A 0 (right) For details, refer to [2,I7].

H


650


cant role. I n the {FeNO}' species the bound NO ligand is more nucleophilic, as in the {FeNO}' species, and thus its protonation to give a H N O species appears reasonable [ 10,193. Transfer of two electrons and two protons will yield the Fe( I I)-hydroxylamine complex. T h e corresponding Fe( 111)-hydroxylamine complex of NiR has been obtained and structurally characterized [lo]. Note that, in model complexes, hydroxylamine adducts can usually be derived only in the Fe( I I) state because of the reducing power of hydroxylamine [12]. T o this point, the initially strong N O bond (nitrite) has been transformed into a single N=O bond (hydroxylamine) at the activesite haem of NiR. It will undergo further reductive cleavage to form the product, ammonia, which dissociates from the active site and will be guided electrostatically to the protein surface through the product outlet channel [8].

histidine. T h e only feature that is strictly conserved between NiR and HAO, apart from the arrangement of the haems [2,8], is a histidine residue at the active site of a topologically conserved helix [2]. In NiR this histidine binds the substrate, and its conservation in the two proteins suggests a role in the transformation nitrite % hydroxylamine.

Mechanism of action of NiR Combining the three-dimensional structures of substrate and intermediate complexes with results from theoretical calculations, a reaction mechanism for NiR has been proposed [lo]; a simplified version is depicted in Scheme 1 [2]. From the resting state, water has to be exchanged for substrate. T h e nitrite ion is bound with its nitrogen atom to the active-site iron. Both spectroscopic and computational studies [5,10,14] show that the reduced form of the active site binds nitrite much more strongly than does the oxidized form. T h e explanation for this is a pronounced back-bonding effect which occurs in the Fe(I1) form but not in the Fe(II1) form. This backbonding interaction, which has also been observed in model complexes [18], is of fundamental importance for the initial steps of the mechanism. Upon binding of nitrite to the active site, the next step involves breaking one of the N-0 bonds of NO;. N-0 bond heterolysis leads to an {FeNO}' species, while homolysis would lead to {FeNO}'. Both reactions were calculated to be energetically possible, but, based on the results obtained for the substrate-binding step, heterolytic cleavage will be supposed. T h e reduction of the {FeNO}' species occurs in two rapid one-electron reductions to form an {FeNOJRspecies. T h e rationale for this lies in the fact that, from the {FeNO}' to the { FeNO}' adduct, a linear-to-bent transition of the Fe-NO unit has to occur. Therefore the highly stable {FeNO}' adduct plays no signifi-

Reaction of NiR with hydroxylamine and derivatives UV/visible spectroscopy represents a suitable tool for initial investigation of the reactivity of multihaem proteins because of their distinct electronic properties and intense absorption maxima in the region 400-650nm. I n the case of NiR, which displays a typical c-type cytochrome spectrum [20], the absorption maxima of the enzyme in the oxidized state [Fe(III), as isolated] are at 280,409 and 536 nm, plus a shoulder at 615 nm that has been assigned to a high-spin Fe(II1) haem centre [5,11,13,14,21,22]. Upon reduction, characteristic maxima appear at 420, 523 and 553 nm, which allow the oxidized and the reduced Fe( I I) states of the enzyme to be distinguished. I n the first set of experiments, reductive titrations with hydroxylamine, its derivatives substituted at both the N- and 0-positions, and hydrazine were carried out in the absence of dioxygen, starting from Fe(II1)-NiR. Hydroxyl-

Scheme I Minimal reaction scheme of NiR, showing the potential bound intermediates nitrogen monoxide and hydroxylamine For a detailed reaction mechanism based on X-ray crystallography, spectroscopy and kinetic measurements, refer to [ 101.

I H

o,N/p 1111

n Fe = I N

I

LYs

II

Ill .N%o

H'

I II

,0-H H2N

2 H' ' r= Fe -f-'=5

3 H'

111

m F e - p I N

I N

I

I

LYS

LYS

65 I

IV H,N

-

I 111

c-5 Fe I N

I

LYS


Biochemical Society Transactions (2002) Volume 30, part 4

amine and derivatives reacted rather poorly (Figure 2), and the maximum reduction of NiR (16 0 4; lo4excess of substrate) was achieved by hydroxylamine and its N-methyl derivative (Table l), compared with approx. 5 o/o for the various N- and 0-substituted derivatives, including hydroxylamine-0-sulphonic acid. This was rather unexpected in view of the results observed for hydroxylamine-iron complexes [12]. On the other hand, hydrazine proved to be a relatively good reductant (67 yo)of NiR under these conditions.

In the set of oxidative titrations, the reactivity of photochemically reduced Fe( I I)-NiR was investigated. Hydroxylamine and the various derivatives, but not hydrazine, did re-oxidize the enzyme very rapidly, with an efficiency of close to 100 Yo. In most cases, the re-oxidation was complete after addition of 5 m M substrate (Table 1, Figure 2). These data support the results obtained previously from soaking experiments with crystalline Fe(II1)-NiR and hydroxylamine, which led to the formation of a stable crystalline Fe( 111)-hydroxyl-

Figure 2 Reaction of NiR with hydroxylamine and derivatives Enzyme was present at 5 ,uM, in I00 mM potassium phosphate buffer, pH 7.0, at 25 "C, in the absence of dioxygen. (A) Oxidative titration of photochemically reduced Fe(ll)-NiR (spectrum I ) with N-t-butylhydroxylamineat 5 mM (spectrum 2), I0 mM (3), 20 mM (4) 30 mM (5), 40 mM (6) and 50 mM (7). Spectrum (8) is that of Fe(lll)-NiR, as isolated. (B) Change in absorbance at 548 nm corresponding to Fe(ll)-NiR after stepwise addition of substrate: solid symbols denote reductive titration of Fe(ll1)-NiR: open symbols denote oxidative titration of photochemically reduced Fe(ll)-NiR. Substrates were hydroxylamine ( 0 ,O), N-methylhydroxylamine (B, u),N-tA),0-methylhydroxylamine(V, V), 0-t-butylhydroxylamine (+, O),hydrazine (4,4) and hydroxylamine-0butylhydroxylamine (A, sulphonic acid (stars).

B

A 0.50

0.08 0.06

m

w

0.04

0.02 0.00 0

10

Wavelength [nm]

20

Substrate

30

[mu]

Table I Reaction of NiR with hydroxylamine and derivatives Enzyme was present at 5 pM, in I00 mM potassium phosphate buffer, pH 7.0, at 25 "C, in the absence of dioxygen. Percentage values were calculated from M,,, after stepwise addition of 50 mM substrate, I 00% = 4

4 ,

for

[Fe(ll)-Nl%hotochem,caliy

reducedl - [Fe(lll)-NIR,,

,solatedl.

Substrate

Fe(ll)-NiR* (%)

Fe(lll)-NiRt (%)

Hydroxylamine N-Methylhydroxylamine N-t-Butylhydroxylamine 0-Methylhydroxylamine 0-t-Butyl hydroxylamine Hydroxylamine-0-sulphonic acid Hydrazine

16 16 5

75 76 78

5

89

6 6 67

94

*Reduction of Fe(ll1)-NiR as isolated 1.Oxidation of photochemically reduced Fe(ll)-NiR.


652

93 20

40

50


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. Usually, the crystals begin to crack. Currently, soaking experiments are under way with Fe(II1)-NiR and several of the 0-substituted hydroxylamine derivatives, in an attempt to obtain a closer look at the binding of hydroxylamine to the active haem centre of NiR.

7 8

9 10

II 12

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

14

15 16

References

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Cole, J. A. and Brown, C. M. ( 1980) FEMS Microbiol. Lett. 7,65-72 Messerschrnidt,A.. Stach, P., Bourenkov, G. P., Einsle. O., Bartunik H. D., Huber, R. and Kroneck, P. M. H. (1999) Nature (London) 400, 476480 Darwin. A.. Hussain. H., Grifiths. L., Grove, J,, Sambongi, Y., Busby, S. and Cole, J. A. ( I 993) Mol. Microbiol. 9, I 255- I 265 Hussain, H.. Grove, J,, Grifiths, L., Busby, 5. and Cole, J. A. ( I 994) Mol. Microbiol. 12, 153-1 63 Barnford, V. A,, Angove, H. C., Seward, H. E.. Thornson, A. J., Cole, J. A., Butt, J. N., Hernmings, A. M. and Richardson, D.J. (2002) Biochemistry 41, 292 1-293 I Fleischrnann, R. D., Adarns, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., Bult, C. J,, Tomb, J,-F.,

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Dougherty, B. A,, Memck J, M. et al. ( I 995) Science 269, 496-5 I2 Simon. J., Gross, R., Einsle, O., Kroneck, P. M. H., Kroger, A. and Klimrnek. 0. (2000) Mol. Microbiol. 35, 686-696 Einsle, O., Stach, P.. Messenchrnidt, A,, Simon, J.. Kroger, A,, Huber, R. and Kroneck P. M. H. (2000) J. Biol. Chern. 275, 39608-396 I 6 Hendrich. M. P., Petasis, D.,Arciero, D. M. and Hooper, A. B. (2001) J. Am. Chem. SOC. 123, 2997-3005 Einsle, O., Messerschrnidt,A,, Huber, R., Kroneck P. M. H. and Neese, F. (2002) J. Am. Chern. SOC., in the press Stach, P., Einsle, O., Schurnacher, W., Kurun, E. and Kroneck, P. M. H. (2000) J. Inorg. Biochem. 79, 38 1-385 Wieghardt, K. ( I 984) Adv. Inorg. Bioinorg. Mechanisms 3, 2 I 3-274 Schumacher, W. and Kroneck, P. M. H. ( I 99 I ) Arch. Microbiol. 156, 70-74 Schurnacher, W., Hole, U. and Kroneck, P. M. H. ( I 994) Biochem. Biophys. Res. Cornrnun. 205,9 I 1-9 I6 Massey, V. and Hernrnerich, P. ( I 977) J. Biol. Chem. 252, 56 12-56 I4 Howes. B. D., Feis, A,, Rairnondi, L., Indiani, C. and Srnulevich. G. (200 I ) J. Biol. Chern. 276, 40704407 I I Igarashi, N., Moriyarna, H., Fujiwara, T., Fukurnori, Y. and Tanaka. N. ( 1997) Nat. Struct. Biol. 4, 276-284 Nasri. H.. Ellison, M. K., Krebs, C., Huynh, B. H. and Scheidt. W. R. (2000) J. Am. Chern. SOC. 122, 10795- I0804 Bonner, F. T. and Peanall, K. A. ( 1982) Inorg. Chern. 2 I , 1973- I978 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

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 Cytochrome haem-copper terized by its the ability to

central to its proposed role in bacterial microaerobic metabolism. Recent spectroscopic characterization of both the cytochrome cbb, oxidase complex from Pseudomonas stutzeri and the dihaem ccOp subunit expressed in Escherichia coli has revealed the presence of a lowspin His/His co-ordinated c-type cytochrome. T h e low midpoint reduction potential of this haem ( E m< 100 mV), together with its unexpected in the reduced state at the ability to bind expense of the distal histidine ligand, raises ques-

ebb, oxidase is a member of the oxidase superfamily. is characwhile retaining high oxygen pump protons. These attributes are

Key words: cytochrorne oxidase, Pseudomonos, respiration, spectroscopy. Abbreviations used: HCO, haern-copper oxidase; SUI, subunit I. 'To whom COrresDondence should be addressed (e-mail r.pitcher(a) uea.ac uk).

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