Eur. J. Biochem. 210,629-633 (1992) (QFEBS 1992
Evidence for two histidine ligands at the diiron site of methane monooxygenase D. Drurnmond S. SMITH and Howard DALTON Department of Biological Sciences, University of Warwick, Coventry, England (Received June 16/September 9,1992)
EJB 92 0846
Circular dichroism spectroscopy has shown the hydroxylase component of methane monooxygenase to have a high helical content. The apoprotein has the same secondary structure as the holoenzyme. Chemical modification shows 12 histidines to be reactive with diethylpyrocarbonate in the holoenzyme, whereas 14 are reactive in the apoenzyme. Two histidine residues are implicated as iron ligands. Further chemical modification results suggest a cysteine residue is in close proximity to the diiron centre.
The soluble methane monooxygenase (MMO) from Methylococcus capsulatus (Bath) catalyses the oxidation of methane to methanol: CH,
+ O 2 + NADH + H + + CH30H + H 2 0 + NAD'.
The enzyme consists of three components all of which have been purified [l -31. The components comprise a reductase [3,4] which contains an FAD and Fe2Sz centre which passes electrons from NADH to the hydroxylase, protein B, a small regulatory protein , and the hydroxylase, which contains a binuclear iron centre . Methane oxidation is believed to occur at the diiron centre of the hydroxylase by a carboncentred radical mechanism [6, 71. The enzymes from three other sources, Methylosinus trichosporium OB3b , Methylobacterium CRL-26 [9, 101 and Methylosinus sporium [ l l ] have also been purified and characterised and are all very similar to the enzyme from M. capsulatus. The bridged diiron centre of the hydroxylase is similar to those found in haemerythrin , rubrerythrin , ribonucleotide reductase B2 protein  and purple acid phosphatases . Crystallographic studies have been performed on haemerythrin [16, 171 and ribonucleotide reductase B2 [I81 and have shown that they are both helical proteins. Five histidine residues comprise the non-bridging ligands in haemerythrin [I21 whereas in ribonucleotide reductase these ligands are two histidine and three acidic residues . Extended X-ray-absorption fine-structure (EXAFS) studies of the hydroxylase component of MMO [I91 show each iron atom is ligated by five or six O/N ligands. Recent electron nuclear double resonance (ENDOR) experiments  suggest there are histidine ligands to the irons in the half-reduced (Fe"/Fe"') form of the hydroxylase from M . trichosporium. Correspondence ro H. Dalton, Department of Biological Sciences, University of Warwick, Coventry, England CV4 7AL Abbreviutions. EXAFS, extended X-ray-absorption fine structure; ENDOR. electron nuclear double resonance; DEPC, diethyl pyrocarbonate; MMO, methane rnonooxygenase. Enzymes. Methane monooxygenase (EC 1.14.1 3.25); ribonucleotide reductase (EC 126.96.36.199).
We have investigated the structure of the hydroxylase by CD spectroscopy and also describe chemical modification experiments suggesting that the diiron centre is ligated by two histidines in the protein from M . capsulatus. MATERIALS AND METHODS The hydroxylase component of MMO was purified by ionexchange and gel-permeation chromatography in a procedure based on that of Woodland and Dalton [l]and was shown to be pure by SDS/PAGE. Enzyme activity was determined at 45 "C in 20 mM Mops buffer, pH 7, b y measuring the production of propylene oxide from propene in the presence of hydroxylase, 5 mM NADH and proteins B and C of the MMO complex essentially as described by Green and Dalton . Protein was determined by the method of Lowry et al.  and iron analyses were performed by chelation with 4,7-diphenyl-1,lo-phenanthroline ( E , 22000 M - ' cm-I). Hydroxylase was depleted of iron by two methods. Incubation with 0.1 M 8-hydroxyquinoline sulphonate removed half of the protein-bound iron giving preparations of hydroxylase containing 1 - 1 .I mol Fe/mol protein. Incubation with 10 mM dihydroxybenzaldehyde followed by ligand exchange using 8-hydroxyquinoline sulphonate allowed apoprotein ( < 0.3 mol Fe/mol protein) to be prepared. Rcconstitition of apohydroxylase was performed by the addition to a final concentration of 1 mM of an anaerobic solution of ferrous ammonium sulphate to an anaerobic preparation of apoprotein (5 mg/ml) containing 20 mM Tris, pH 7.5, and 5 mM dithiothreitol. After incubation under anaerobic conditions at 20°C for 15 min air was admitted to the reaction and incubation continued on ice for a further 150 min prior to assay. CD spectra were recorded at 25°C using a Jasco model 760 spectropolarimeter using a 0.1-cm pathlength cell and accumulating 10 scans/spectrum. Samples for CD measurements were prepared in 10 mM potassium phosphate, pH 7, the protein concentrations were 0.08 mg/ml for scans of the
630 far-ultraviolet region and 3.5 mg/ml for scans in the near ultraviolet. Chemical modification of hydroxylase with diethylpyrocarbonate (DEPC) was performed at 25°C in 50 mM sodium phosphate, pH 7. The reaction was monitored spectrophotometrically and the number of histidine residues modified quantified using the absorbance increase at 242 nm during the reaction due to the formation of carbethoxyhistidine (e, 3200 M - ' cm-') . The reaction of hydroxylase with 2-nitro-5-thiocyanobenzoic acid was performed in 50 mM Hepes, pH 8, and monitored by observing the production of 2-nitro-5-thiobenzoic acid at 412 nm (e, 13600 M-' cm-').
RESULTS DEPC modification of protein A
Histidine residues are known to be ligands to the diiron centres of haemerythrin  and of ribonucleotide reductase . Histidine is therefore a likely candidate for ligation of the binuclear iron centre of MMO hydroxylase. In the absence of structural changes between holoenzyme and apoenzyme a difference in the number of histidine residues rcacting with DEPC in holoenzyme and in iron-depleted forms of the enzyme would reflect the number of histidines protected from reaction by the iron atoms; these residues would most probably be ligated to the binuclear iron centre. Several lines of evidence suggest apoenzyme and holoenzyme have the same protein structure, The apoenzyme of MMO hydroxylase has the same molecular mass as holoenzyme, determined by HPGPC, thus the iron centre is not important in the retention of the protein quaternary structurc. Holocnzyme and apoenzyme elute from anion-exchange columns at the same ionic strength, and CD spectroscopy shows apoenzyme to have a similar structure to holoenzyme. In addition to this evidence that these forms of the protein have the same structure, it is also possible to recover catalytic activity from apoenzyme following reconstitution of the diiron centre using ferrous ammonium sulphate and dithiothreitol. Apoenzyme itself is totally inactive, reconstituted material has up to 40% of the activity of untreated holoenzyme. Chemical modification of MMO hydroxylase holoenzyme with DEPC was studied by difference spectroscopy. During the course of the reaction the absorbance at 240 nm increased duc to the formation of carbethoxyhistidine, no change in the absorbance at 280 nm was observed, indicating that in this form of the protein tyrosine residues did not react with DEPC. Reaction of holoenzyme with this reagent did not change the iron content of the protein. After completion of the modification reaction approximately 12 mol histidine/mol protein (11.8 f 0.7, n = 6) had been modified (Fig. 1). Further sddition of reagent did not increase the extent of histidine modification. The catalytic activity of hydroxylase and 1Fe-A toward the epoxidation of propylene was tested using an assay involving electron transfer from NADH mediated by the other components of the MMO complex (proteins B and C). 1Fe-A had 60 - 90% of the activity of holoenzyme. Extcnsive reaction with DEPC (such that 9 mol histidine/mol protein were modified) caused inactivation. This suggests that histidine residues may be involved in the interaction of the hydroxylase with other components of the MMO complex and/or in electron transfer within the hydroxylase component but the possible
Fig. 1. Time course of the reaction of MMO hydroxylase with 500 pM DEPC, ( 0 )holoenzyme and (0) 1Fe-A (iron-depleted hydroxylase).
modification of other potentially important amino acids (e. g. lysine residues) cannot be excluded. The time course of the reaction of DEPC with 1 Fe-A (a form of the hydroxylase containing 50% of the normal complement of iron) is shown in Fig. 1. Again, addition of more reagent did not increase the extent of reaction, nor was reaction with tyrosine observed. In this case approximately 14 mol histidine/mol protein were modified (14.0 +_ 0.6, n = 5). Two histidine residues which were unreactive in holoenzyme become reactive upon iron depletion and so may be implicated as being iron ligands. A direct comparison of the modification of apoenzyme with that of holoenzyme was not possible since reaction of DEPC with apoenzyme (but not with holoenzyme or 1 Fe-A) led to some reaction with tyrosine residues, seen as a decrease in absorbance at 280 nm during reaction. Reaction of DEPC with tyrosine precludes the accurate estimation of hstidine modification in apoenzyme since it also affects the absorbance in the 240-nm region . In order to compare the extent of modification of histidine residues in apoenzyme with that in other forms of the hydroxylase it was therefore necessary to block the reactive tyrosine residues prior to DEPC niodification. This was possible by reaction with N-acetylimidazolc. It was then posyible to compare the modification of acetylated apoenzyme with that of acetylated holoenzyme without interference from tyrosine modification. It was found that 1.5 additional histidine residues were reactive in acetylated apoeniyme. Apo hydroxylase contains some residual iron ( M 0.3 mol/mol protein). If, as seems likely. this iron is present as intact diiron centres then the difference in the extent of modification of apo-forms and holo-rorms of the protein may be underestimated by approximately 15% and the measured value of 1.5 residues may reflect a true value nearer 1.7 residues, strongly supporting ligation of each diiron centre by two histidine residues. In a series of measurements of histidine modification in holoenzyme and in 1Fe-A. 2.2 0.4 more residues were found to be reactive in IFe-A. This i s good evidence for iron ligation by two histidines and is supported by the results of DEPC modification of acetylated apoenzyme and holoenzymes. Chemical modification with 2-nitro-5-thiocyanobenzoic acid The time-courses for the release of the 2-nitro-5-thiobenzoate anion during the reaction of hydroxylase holoen-
Fig. 2. Timc course of the reaction of MMO hydroxylase with 500 pM 2-nitro-5-thiocyanobenzoic acid at 25OC, ( 0 )holoenzymc and (0) apoenzyme. TNR, 5-thio-2-nitrohen7oate.
zyme and apoenzyme with the cysteine-modifying reagent 2nitro-5-thiocyanobenzoicacid are shown in Fig. 2. Reaction of holoenzyme with 2-nitro-5-thiocyanobenzoicacid leads to the cyanylation of 0.65 mol cysteinejmol protein whereas reaction of apoenzyme leads to cyanylation of 1.27 residues/molecule. The rates of rcaction of apoenzyme and holoenzyme with 2-nitro-5-thiocyanobenzoic acid are very similar, being approximately tenfold slower than the rate of reaction of free cysteine. It is very unlikely that there is direct ligation of cysteine residues with the diiron centre since no charge-transfcr bands are found in the spectrum of the protein. The additional residue modified in the apoenzyme is probably close to the iron centre and in the holoenzyme is protected by it from reaction with 2-nitro-5-thiocyanobenzoicacid. The results of reactions of holocnzyme and apoenzymc forms of MMO hydroxylase with 5,5’-dithiobis-(2-nitrobenzoic acid) are similar to those obtained using 2-nitro-5thiocyanobenzoic acid titration. 5,5’-dithiobis-(2-nitrobenzoic acid) modifies 2.8 residues in holoenzyme and 3.6 rcsidues in apoenzyme again suggesting a cysteine residue is protected from reaction by the diiron centre.
CD spectroscopy In order to support the conclusions reached using chemical modification, the structure of the MMO hydroxylase was investigatcd using CD spectroscopy. We have performed CD measurements on MMO hydroxylasc holoenzyine (containing 2 mol Fe/moI protein) and apoenzyme in order to determine the secondary structural elements present in the protein and to investigate whether iron removal causes significant structural perturbations within the enzyme. CD signals were obtained in the far-ultraviolct (Fig. 3) and near-ultraviolet (Fig. 4) regions of the spectrum from both holoenzyme and apoen7yme. Negative CD bands at 208 nm and 222 nm accompanied by a positive signal at 192 nm characteristic of the CD spectra of a-helices are clearly seen in the far-ultraviolet CD spectra of both holoenzyme and apoenzyme. Analysis of the far-ultraviolet region of the spectrum of holoenzyme by reference to spectra of proteins of known structure  shows it to be comprised of around 45% a-helical structure. The spectrum of apoenzyme is very similar to that of holoenzyme, an esti-
220 wavelength (nrn)
Fig. 3. Far-ultraviolet CD spectra of MMO hydroxylase holoenzymc and apoenzyme.
305 wavelength (nm)
Fig. 4. Near-ultraviolet CD spectra of MMO hydroxylase, (A) holoenzyme, (B) apoenzyme.
mate of 40% helix being obtained for this form of the protein. This is not significantly different from the helix content of the holoenzyme. The MMO hydroxylase contains a high proportion of helical structure, in common with other binucleariron-containing proteins [I 6 - 1XI, and iron removal does little to disturb the secondary structure of the protein, as evidenced by the similarity of the far-ultraviolet CD spectra of holoenzyme and apoenzyme. We have also obtained the far-ultraviolet CD spectrum of ribonucleotide reductase protein €32. X-ray crystallography yields an estimate of the helical content of this protein of approximately 70% [IS]; the helical nature of the protein is clearly seen in the CD spectrum (Fig. 5). Analysis of the spectrum gives an estimate of the helicity of the protein of approximately 54%, in reasonable agreement with the crystallographic data. A very low amount of p-structure is suggested by CD, also in agreement with the X-ray structure in which only one region of j-sheet was found . If the helical content of MMO hydroxylase is calculated using parameters derived from the CD spectrum of ribonucleotide reductase and the crystallographic estimate of the helicity of that protein (70%) then an estimate of 58% helix is obtained both for hydroxylase holoenzyme and apoenzyme. CD signals observed in the near-ultraviolct region arc due to assymetries in the environments of aromatic residues and
Fig. 6. Proposed structure of the active site of MMO hydroxylasc.
220 wavelength (nrn)
Fig. 5. Far-ultraviolet CD spectrum of ribonucleotide-reductaseprotein B2.
are sensitive indicators of structural changes around the residues contributing to the CD spectrum. There is some difference in the CD-spectra Of h0l0 hydroxylasc and apo-hydroxYlase in this region (Fig. 4) suggesting that although the secondary structure of the protein is largely unaffected followin&iron removal some, possibly localised, structural changes do occur.
. This model (Fig. 6) contains two ligating histidine residues which is consistent with our experimental observations. At the equivalent position to that of the tyrosyl radical present in ribonucleotide reductase, in close proximity to the diiron centre, molecular-modelling studies suggest the presence of a cysteine residue in MMO . This residue may be that which is reactive in MMO anoenzvme but not in holoenzyme, we intend to test this hypothesis by isolating and a peptide containing the modified cysteine residue. We thank Dr s. E. Radford, Ulliversity of Oxford, England for assistance with the CD spectroscopy and Dr Marc Fontecave, Universite Joseph Fourier, Grenoble, France for the gift of ribonucleotide reductase. This work was supported by the Gas Research Institute, Chicago, IL, USA under contract no. 5089-260-1826.
REFERENCES DISCUSSION Using CD spectroscopy we have shown that the hydroxylase component of MMO contains a high degree of helical structure. Ribonucleotide reductase protein B2 [I 81 and haemerythrin [16,17]are both largely comprised of helical structure. The amino-acid ligands at the diiron sites of ribonucleotide reductase and of haemerythrin are very different and X-ray crystallography studies [18,26, 271 have shown that the disposition of the diiron centres in these proteins with respect to the helix axes is different, nonetheless in both cases the amino-acid ligands are contained in helices. The same may be true of the MMO active-site amino acids. MMO hydroxylase contains a total of 52 histidine residues. The chemical modification results above, using DEPC to modify histidine residues in different forms of MMO hydroxylase, strongly suggest that two of these histidine residues act as ligands to the diiron centre. Ligation of the iron centre by two histidines is certainly consistent with EXAFS studies of the protein . The presence of two histidine ligands at the active site of MMO suggests it is more similar to that of ribonucleotide reductase protein B2 than that of haemerythrin which contains five histidine ligands. There is some sequence similarity between MMO and ribonucleotide reductase  which occurs in the region of those amino acids which ligate the diiron centre of ribonucleotide reductase. Recently the sequence of rubrerythrin has also been found to be similar to the same region of the MMO sequence . The active site of ribonucleotide reductase contains two histidine residues, one bonding to each iron atom .Using the sequence similarity between MMO hydroxylase and ribonucleotide reductase and the crystallographic structure determined for ribonucleotide reductase [l8] a model for the structure of the diiron site of- MMO has been proposed
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