Resonance Raman spectroscopic characterization of the nickel cofactor, F430, form methanogenic bacteria

Biochimica et Biophysica Acta, 748 (1983) 143-147

143

Elsevier

BBA 31718

RESONANCE RAMAN SPECTROSCOPIC CHARACTERIZATION OF THE NICKEL COFACTOR, F430, F R O M METHANOGENIC BACTERIA A N D R E W K. S H I E M K E a, L. D U D L E Y EIRICH b and T H O M A S M. L O E H R a,,

a Department of Chemistry and Biochemical Sciences, Oregon Graduate Center, 19600 N.W. Walker Road, Beaverton, OR 97006, and I, Department of Biology, Portland State University, Portland, OR 97207 (U.S.A.) (Received May 4th, 1983)

Key words: Cofactor F43o; Ni complex," Resonance Raman spectroscopy; Methanogenesis; (Methanobacterium)

Resonance Raman spectroscopy was used to investigate the Ni-tetrapyrrole, cofactor 17430, of methanogenic bacteria. Excitation within the 430-nm absorption band produced resonance-enhanced vibrational modes and a spectral pattern that contrasts with resonance Raman data of other metal-tetrapyrrole complexes (heme-, metalloporphyrin- and vitamin B-12-derivatives). With 406.7-nm excitation, the most intense spectral features were observed at 1530, 1628 and 1562 cm-i. Although many weaker spectral bands were recorded below 1500 cm-1, the relative simplicity of the F430 resonance Raman spectrum is in accord with the lower degree of unsaturation in the nickel complex. Excitation at longer wavelengths brought several low-frequency modes in the 200-500 cm -! region into resonance. This observation indicates that the 430-nm band is composed of multiple electronic transitions. The low-frequency spectrum, which should contain metal-ligand vibrations, may be particularly useful in studying the interactions of F430 with coenzyme M and methylreductase.

1. Introduction I=43o, the low molecular weight cofactor of the methylreductase of methanogenic bacteria [1], is the first naturally occurring nickel complex to be structurally characterized [2]. Furthermore, this complex is an Ni-derivative of a novel tetrapyrrole [2]. Biosynthetic studies have revealed that the macrocyclic ligand is synthesized from eight molecules of 8-aminolevulinic acid and incorporates two methionine-derived methyl groups [3,4]; these observations suggested that F43o should be placed under the sirohydrochlorins, along with sirohemes and vitamin B-12, rather than among iron porphyrins and chlorophylls. Diekert et al. [5] had pointed out that the

* To whom correspondence should be addressed 0167-4838/83/$03.00 © 1983 Elsevier Science Publishers B.V.

optical absorption spectrum of F43o was reminiscent of the corrinoids. The recent structural analysis (albeit of a methanolysis product of the cofactor, F430 M) has established the correctness of that suggestion insofar as the ligand skeleton is a tetrahydro-corphin derivative [2]. Of all natural tetrapyrrole macrocycles, this new F430 chromophore is the most highly reduced one known so far (Fig. 1). The parent compounds of F430 ( M r estimated at approx. 1500 per gatom Ni) have an intense visible absorption band at 430 nm (cNi = 23000 M -1. cm -1) and a second prominent band at 274 nm (¢~i = 20000 M -1. cm -1) [2,4,6]. Different preparations of F43o show varying amounts of low-intensity absorption above 500 nm. In the circular dichroism spectrum, however, distinct features with negative ellipticities are observed at approx. 510 nm and approx. 590 nm [2].

144 o

Heme

F4~0

13-12

Fig. 1. The principal chromophore structures of naturally occurring tetrapyrrole complexes: Left: the iron-porphyrin nucleus of heme proteins. Center: the novel tetrahydrocorphin nucleus of the Ni-cofactor, F430. Right: the corrin nucleus of the cobalamin (vitamin B-12) cofactor. Side-chains and axial ligands have been omitted.

In the present communication we report the first application of resonance Raman spectroscopy to the characterization of this novel Ni-chromophore. Our data show that the resonance Raman spectra of F430, while reminiscent of metalloporphyrins and cobalamins, are, however, unique to this new structure, and that Raman spectroscopy should be useful in elucidating the electronic and molecular structure of this complex and its alterations upon protein binding.

(40000 × g, 15 min), the solution was chromatographed on QAE-Sephadex A-25 (2.5 × 30 cm, bicarbonate form) which had previously been equilibrated with distilled water. Elution was performed with a 1500 ml gradient (0 to 0.3 M) of (NH4)HCO 3. The yellow fractions were pooled and chromatographed on Sephadex G-25 (2.5 × 100 cm) which had been equilibrated with 0.1 M (NH4)HCO 3 (pH 7.5). Elution was performed with the same buffer. Further purification of the yellow fractions was accomplished on a Sephadex G-15 column, under conditions identical to that of the Sephadex G-25 column. The final product, F430(M./.), had an ultraviolet/visible spectrum identical to those published for F430b [6] and F430II

[7]. Resonance Raman spectra were collected on an automated Jarrell-Ash spectrophotometer described previously [8]. Samples of F430 were contained in glass capillaries and their spectra were recorded at room temperature using SpectraPhysics Model 164 Ar and Kr ion lasers and a 90 ° scattering geometry. The scattered light was measured by a cooled photomultiplier (RCA C31034) and photon-counting electronics (ORTEC 9302). Signal-to-noise was enhanced by repetitive scanning. 3. Results and Discussion

2. Experimental The two samples of F43o used in the present study were isolated from Methanobacterium bryantii and M. thermoautotrophicum, respectively. The sample from M. bryantii, F430(M.b.), was a generous gift from Drs. W.B. Whitman and R.S. Wolfe. It has been previously described as F430b with an c430=2.3 • 10 4 M -1 .cm -1 and an approximate molecular weight of 1560 per gatom of Ni [6], or as the principal purified fraction F430 (also F430II) which has served as the starting material in further studies [71. Cells (200 g wet weight) of M. thermoautotrophicum were disrupted in a French pressure cell at 20 000 lb/inch 2 in 0.05 M potassium phosphate buffer, pH 7.0. The resulting lysate was heated at 95°C for 10 min and returned to room temperature for all subsequent treatments. Following centrifugation of debris and denatured proteins

Variations in the method of isolation and purification of F430 yield different chemical derivatives of the cofactor. Thus, different F430 parent compounds have been obtained by high temperature or acid treatment of cells to release protein-bound cofactors, followed by extensive column chromatography [6,7,9]. Additional treatment, including modification with toluenesulfonic acid in anhydrous methanol yields F430M, the methanolysis product whose structure has been reported [2]. Although the principal absorption bands at 430 and 274 nm are comparable in these species, F430M differs from F430 parent compounds in that its molecular weight of approx. 975 indicates a loss of some 500-600 mass units upon methanolysis and its optical and CD spectra show a considerable loss of intensity at 550-600 nm [2]. Substances which have been identified as contributing to the extra mass of the 1::430parent compounds are amino

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FREQUENCY, CM - I Fig. 2. ResonanceRaman spectrum of 1::430 isolated from Methanobacteriumb~.antii (A430 = 2.2) using 406.7-nm excitation(24 mW). The peak at 981 cm-1 is due to 0.05 M SO~- added as an internal frequencyand intensity standard. Spectrometerconditions: scan rate 1 cm- l/s; slits 8 cm - 1; scan accumulations: 3 × high-frequency, 5 × low-frequencyregion.

acids, carbohydrates [10] and, in certain cases, coenzyme M (HS-CH2-CH2-SO3-) [91. The additional components in the F430 parent compounds may provide axial ligation for the nickel ion. The methanolysis macrocycle, F430M, is diamagnetic [2] and can be assigned to low-spin Ni(II) in a square planar environment. The parent compounds probably also contain divalent nickel. F430(M.b.), for example, only yields an EPR-detectable Ni(III)-type spectrum after oxidation with hexachloroiridate (J. Lancaster, personal communication). In addition, the F430 parent compounds seem to undergo rather facile changes in spin state [9]. The ability for Ni(II) to vary between high-spin and low-spin forms is more characteristic of square pyramidal or octahedral than square planar coordination and in the case of F430 would correspond to the nickel macrocycle with one or two axial ligands, respectively. The actual spin states of the two samples used in this investigation, F43o(M.b.) and F43o(M.t.), have not been rigorously established. A preliminary 90 MHz IHN M R spectrum of a 6 mg sample of F43o(M.t.) revealed none of the resonances of the corphin macrocycle which were reported for F430M [2]; this may indicate that the sample is paramagnetic, as reported for pMF430 [9].

The resonance Raman spectrum of F430(M.b.) obtained with 406.7-nm excitation is shown in Fig. 2. This spectrum is dominated by a peak at 1530 cm -1, and it is accompanied by two peaks of approximately half its intensity at 1628 and 1562 cm-1; weaker features were observed at approx. 1384, 1300, 1238, 1176, 1080, 905, 755, 715, 637 and 316 cm -1. Factor F43o(M.t. ) yielded a Raman spectrum essentially identical to that of F430(M. b.). The low-frequency region of the spectrum of F43o(M.t. ) is shown in Fig. 3 (upper spectrum). Raman spectral intensities were sensitive to excitation wavelength, as expected for resonanceenhancement arising from electronic transitions within the 430-nm absorption band. In both absorption and circular dichroism spectra, the 430nm band is broad and appears to contain at least one shoulder on the high energy side (approx. 410 nm) of the main 430-nm peak. The existence of multiple electronic transitions is confirmed by our Raman experiments with the available excitation wavelengths of 406.7, 457.9, 476.5 and 488.0 nm. The strong Raman peaks at 1628, 1562 and 1530 cm -1 showed 2-3-times higher intensities with excitation at 406.7 nm than at 457.9 nm, and the intensities decreased further with longer excitation wavelengths. Since 406.7 and 457.9 nm are ap-

146 I 4 0 6 . 7 nm

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proximately equidistant in energy from the 430-nm absorption maximum, the additional resonance enhancement with 406.7-nm excitation must be due mainly to the participation of the approx. 410-nm electronic transition. Although most of the weaker R a m a n peaks showed the same enhancement behavior as the three strong R a m a n peaks, a strikingly different behavior was observed for a number of the lowfrequency vibrations. As can be seen in Fig. 3, with 457.9-nm excitation the peaks at 637, 319 and 185 cm -1 have increased markedly in intensity relative to the 981 cm -1 sulfate standard in that sample. Furthermore, features at 547, 493, 435, 385 and 255 cm -1 have now appeared which were not resolved with 406.7-nm excitation. The enhancement profile for these vibrational modes implies a contribution from an additional electronic transition at slightly lower energy than 430 nm. A preliminary interpretation of the resonance R a m a n spectrum of F430 is best done by reference to the extensively studied spectra of hemes [11,12] and corrins [13,14]. For this comparison (Fig. 4), we selected the divalent, paramagnetic metal complexes, deoxyhemoglobin and reduced cobalamin,

I

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II ,, ,Ill

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Fig. 3. Resonance Raman spectra (low-frequency region) of F430 isolated from Methanobacterium thermoautotrophicum (A430 ~1.4) using 406.7-nm (upper spectrum) and 457.9-nm (lower spectrum) excitation (approx. 35 mW). Spectrometer conditions: as in Fig. 1, but 15 scan accumulations for each spectrum followed by a 25-point smooth. The broad feature at 400-500 c m - 1 in the upper spectrum is due to Raman scattering from the glass capillary. The [SO2 - ] is considerably lower in this sample than for that shown in Fig. 2.

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Fig. 4. Correlation chart of resonance Raman frequencies and intensities of divalent metalloporphinoid complexes: deoxyhemoglobin, 514.5-nm ( > 600 cm-1) and 457.9-nm ( < 600 cm- ]) excitation, from Refs. 15-18; reduced cobalamin(B-12r), 488.0-nm excitation, from Refs. 13 and 14; and factor Fa30' 406.7-nm excitation, from Fig. 2. The spin state for the Ni(lI) in these samples has not been unambiguouslyestablished.

and chose R a m a n spectra which had been generated by visible excitation. The visible absorption bands in these complexes are assigned primarily to ~r ~ ~r* transitions in the conjugated portion of the macrocycle [19,20] and, thus, only those vibrations involving the unsaturated portion of the ligand and the associated metal center are expected to be resonance-enhanced. The R a m a n spectral patterns reproduced in Fig. 4 show that each of these macrocyclic complexes gives rise to a wealth of resonance-enhanced vibrational modes. Furthermore, for heme systems it has been found that certain R a m a n frequencies and intensities are sensitive indicators of the oxidation state and spin state of the coordinated metal ion [11,12]. Corrin R a m a n spectra are also affected by changes in metal oxidation state [13]. In the deoxyhemoglobin spectrum (Fig. 4) the peaks of highest frequency and intensity are due to C=C and C=N stretching vibrations and are found in the approx. 1200-1600-cm -1 range. The overall simplification of the spectral pattern in this region

147

on going from the heme to the corrin to the F430 system correlates well with the decreasing number of unsaturated bonds and decreased extent of conjugation of the ~r-system. The hemes, unlike the corrins and F430, have unsaturation associated with the outer pyrrole carbons and their stretching vibrational modes have been assigned to the highest energy intense feature (e.g., 1607-cm -1 peak in deoxyhemoglobin) [16]. No such vibrations should appear in the corrin and F430 spectra. It is, therefore, tempting to assign the 1628 cm -1 feature in F430 to the vibration of the conjugated C=O and the two remaining 1530 and 1560 cm -1 peaks to the vibrations of the conjugated C=C and C=N network. The low-frequency region of the tetrapyrrole resonance Raman spectra contains principally macrocyclic ring deformation modes and metalligand modes [12,21]. All of the systems in Fig. 4, for example, exhibit two or three peaks in the 600-800-cm-1 region. Below 500 cm-1, the contributions of in-plane M-N and axial M-ligand modes should be observed. Since the low-frequency vibrations of F430 were particularly enhanced with 457.9-nm excitation, these spectra may provide information on axial ligands to the nickel ion in different F430 preparations and derivatives. This is of interest because substrate interactions with macrocyclic metal cofactors generally occur at the axial position. Recently, Keltjens et al. [22] have identified 6,7-dimethyl-8-ribityl-5,6,7,8-tetrahydrolumazine as a possible constituent of F430 which may well be ligated to the nickel ion via the N-5 of the lumazine derivative. Thus, resonance Raman spectroscopy should prove to be a valuable spectroscopic technique for investigating the structure of F430 in isolated cofactors as well as in intact metalloenzymes.

Acknowledgements The authors thank Professor Joann SandersLoehr for helpful advice, particularly with the critical reading of this manuscript. We are grateful to Dr. William B. Whitman and Professor Ralph S. Wolfe for generously supplying a purified specimen of F430(M.b.), to Dr. Peter Lammers for contributing to the initial phase of this investigation, and to Dr. Jack Lancaster for sharing unpub-

lished results. This research was made possible by grants from the National Institutes of Health (GM 18865) and the National Science Foundation (PCM 8011631).

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