Coordination chemistry of F430. Axial ligation equilibrium between square-planar and bis-aquo species in aqueous solution

X-ray absorption spectroscopic characterization of axial ligand coordination to factor F430, the nickel-tetrapyrrole cofactor of the S-methyl-coenzyme M (CH3SCoM) methyl reductase enzyme from methanogenic bacteria, is presented. The nickel of isolated F430 is hexacoordinate at 10 K in aqueous solution (as is the enzyme-bound cofactor), whereas the epimerized and ring-oxidized derivatives of F430 have four-coordinate nickel. Reduction of the ring-oxidized derivative, F560, with dithionite yields F430 in its native configuration, with axial ligands indistinguishable from those present when the cofactor is obtained directly from the holoenzyme. Thus, we conclude that the axial ligands to F430 in aqueous solution are water molecules. Analysis of the nickel extended x-ray absorption fine structure is consistent with this conclusion. Resonance Raman spectra obtained at room temperature contain features characteristic of both 4- and 6-coordinate forms of the cofactor. We have found that the resonance Raman, optical, and x-ray absorption spectra of aqueous solutions of F430 are temperature-dependent due to a ligand-binding equilibrium involving the square-planar and 6-coordinate bis-aquo forms of the cofactor. At low temperatures (less than 250 K) the 6-coordinate form predominates, whereas higher temperature solutions contain both 4- and 6-coordinate species in a dynamic equilibrium. Similar behavior is observed in other weakly coordinating solvents such as methanol and ethanol. The 4-coordinate form is predominant in solvents with strong electron-withdrawing substituents such as 2,2,2-trifluoroethanol and 2-mercaptoethanol. The relevance of this facile ligand exchange to the active site structure and enzymatic mechanism of the parent enzyme is discussed.     

Structural heterogeneity and purification of protein-free F430 from the cytoplasm of Methanobacterium thermoautotrophicum

F430 is the nickel containing tetrapyrrole cofactor of S-methyl coenzyme M methylreductase, the enzyme that catalyzes the final step of methane production by methanogenic bacteria: the reduction of S-methyl coenzyme M (H3CSCH2CH2SO3-) to methane and coenzyme M (HSCH2CH2SO3-). The protein-free F430 obtained from the cytosol of Methanobacterium thermoautotrophicum, strain delta H, exists predominantly in two isomeric forms that differ in relative stereochemical disposition of acid side chains at the 12 and 13 positions of the macrocycle periphery (Pfaltz, A., Livingston, D. A., Jaun, B., Diekert, G., Thauer, R. K., and Eschenmoser, A. (1985) Helv. Chim. Acta 68, 1338-1358). A simple one-step chromatographic procedure for the large-scale separation of these isomers is described. X-ray absorption spectroscopic studies show that F430 (i.e. the native isomer) is 6-coordinate with long nickel-ligand bonds (approximately 2.1 A), suggesting an approximately planar macrocycle. In contrast, the 12,13-diepimer exhibits a 4-coordinate, square-planar structure with short nickel-nitrogen bonds (approximately 1.9 A), suggesting a ruffled macrocycle. Previous reports, based on other x-ray absorption spectroscopic data, of static disorder in F430 Ni-N distances are shown to be incorrect due to sample heterogeneity. The optical spectrum of F430 (whether purified from the protein-free cytosol or extracted at high ionic strength from the holoenzyme) differs significantly from that of the 12,13-diepimer. The optical spectral differences are correlated with the alterations in coordination number and geometry of the central nickel ion in the two F430 isomers.     

The magnetic and electronic properties of Methanobacterium thermoautotrophicum (strain delta H) methyl coenzyme M reductase and its nickel tetrapyrrole cofactor F430. A low temperature magnetic circular dichroism study

Variable temperature magnetic circular dichroism (MCD) spectroscopy has been used to characterize the magnetic and electronic properties of the Ni(II) tetrapyrrole, F430, which is the cofactor of the S-methyl coenzyme M methylreductase enzyme from Methanobacterium thermoautotrophicum (strain delta H). 4-Coordinate forms are found to be diamagnetic (S = 0 ground state), whereas 6-coordinate forms are paramagnetic (S = 1 ground state). MCD studies, together with parallel low temperature UV-visible absorption and resonance Raman investigations, show that the equilibrium distribution of 4-coordinate square-planar and 6-coordinate bis-aquo forms of the native isomer of F430 in aqueous solution is affected by both temperature and the presence of glycerol. In the presence of 50% glycerol, the 12,13-diepimer of F430 is shown to be partially 6-coordinate in frozen solution at low temperature. Low temperature MCD magnetization data allow the determination of the axial zero-field splitting (D) of the S = 1 ground state of bis-ligand complexes of F430. The value of D is sensitive to the nature of the Ni(II) axial ligands: bis-aquo F430, D = +9 +/- 1 cm-1; bis-imidazole F430, D = -8 +/- 2 cm-1. Measurement of D = +10 +/- 1 cm-1 for F430 in the methylreductase holoenzyme argues strongly against histidine imidazole coordination to Ni(II) in the enzyme. The possible existence of alcoholic or phenolic oxygen-containing ligands (serine, threonine, tyrosine, water) to Ni(II) in the enzyme-bound cofactor is discussed.     

Coordination and geometry of the nickel atom in active methyl-coenzyme M reductase from Methanothermobacter marburgensis as detected by X-ray absorption spectroscopy

Methyl-coenzyme M reductase (MCR) catalyzes the reduction of methyl-coenzyme M (CH(3)-S-CoM) to methane. The enzyme contains as a prosthetic group the nickel porphinoid F(430) which in the active enzyme is in the EPR-detectable Ni(I) oxidation state. Crystal structures of several inactive Ni(II) forms of the enzyme but not of the active Ni(I) form have been reported. To obtain structural information on the active enzyme-substrate complex we have now acquired X-ray absorption spectra of active MCR in the presence of either CH(3)-S-CoM or the substrate analog coenzyme M (HS-CoM). For both MCR complexes the results are indicative of the presence of a five-coordinate Ni(I), the five ligands assigned as four nitrogen ligands from F(430) and one oxygen ligand. Analysis of the spectra did not require the presence of a sulfur ligand indicating that CH(3)-S-CoM and HS-CoM were not coordinated via their sulfur atom to nickel in detectable amounts. As a control, X-ray absorption spectra were evaluated of three enzymatically inactive MCR forms, MCR-silent, MCR-ox1-silent and MCR-ox1, in which the nickel is known to be six-coordinate. Comparison of the edge position of the X-ray absorption spectra revealed that the Ni(I) in the active enzyme is more reduced than the Ni in the two EPR-silent Ni(II) states. Surprisingly, the edge position of the EPR-active MCR-ox1 state was found to be the same as that of the two silent states indicating similar electron density on the nickel.     

Nonionic amphiphilic polymers derived from Tris(hydroxymethyl)-acrylamidomethane keep membrane proteins soluble and native in the absence of detergent

The UV-visible, circular dichroism (CD), and resonance Raman (RR) spectra of the wild type yeast iso-1-cytochrome c (WT) and its mutant F82H in which phenylalanine-82 (Phe-82) is substituted with His are measured and compared for oxidized and reduced forms. The CD spectra in the intrinsic and Soret spectral region, as well as RR spectra in high, middle, and low frequency regions, are discussed. From the analysis of the spectra, it is determined that in the oxidized F82H the two axial ligands to the heme iron are His-18 and His-82 whereas in the reduced form the sixth ligand switches from His-82 to Met-80 providing the coordination geometry similar to that of WT. Based on the spectroscopic data, the conclusion is that the porphyrin macrocycle is less distorted in the oxidized F82H compared to the oxidized WT. Similar distortions are present in the reduced form of the proteins. Frequency shifts of Raman bands, as well as the decrease of the alpha-helix content in the CD spectra, indicate more open conformation of the protein around the heme.     

Characterization of the heme environmental structure of cytoglobin,a fourth globin in humans

Cytoglobin (Cgb) represents a fourth member of the globin superfamily in mammals, but its function is unknown. Site-directed mutagenesis, in which six histidine residues were replaced with alanine, was carried out, and the results indicate that the imidazoles of His81 (E7) and His113 (F8) bind to the heme iron as axial ligands in the hexacoordinate and the low-spin state. The optical absorption, resonance Raman, and IR spectral results are consistent with this conclusion. The redox potential measurements revealed an E' of 20 mV (vs NHE) in the ferric/ferrous couple, indicating that the imidazole ligands of His81 and His113 are electronically neutral. On the basis of the nu(Fe-CO) and nu(C-O) values in the resonance Raman and infrared spectra of the ferrous-CO complexes of Cgb and its mutants, it was found that CO binds to the ferrous iron after the His81 imidazole is dissociated, and three conformers are present in the resultant CO coordination structure. Two are in closed conformations of the heme pocket, in which the bound CO ligand interacts with the dissociated His81 imidazole, while the third is in an open conformation. The nu(Fe-O2) in the resonance Raman spectra of oxy Cgb can be observed at 572 cm(-1), suggesting a polar heme environment. These structural properties of the heme pocket of Cgb are discussed with respect to its proposed in vivo oxygen storage function.     

Is coenzyme M bound to factor F430 in methanogenic bacteria? Experiments with Methanobrevibacter ruminantium

Coenzyme M (2-mercaptoethane sulfonic acid) and factor F430 (a nickel porphinoid) are coenzymes found in methanogenic bacteria. Recently it has been proposed that in these bacteria a coenzyme MF430 also exists which plays a key role in methane formation and in which coenzyme M and F430 are bound to each other. To test this hypothesis Methanobrevibacter ruminantium, which requires coenzyme M as a vitamin, was grown in the presence of [2-14C]CoMSH. F430 and 'CoM' (mixture of CoMSH and its disulfides) were quantitatively extracted from these cells and from partially purified methyl-CoM reductase using various methods. The extracts were chromatographed on cellulose or Sephadex G-10. Under all conditions factor F430 and 'CoM' were completely (greater than 99%) separated. There was no indication for the existence of a protein-free F430 species containing covalently bound coenzyme M in Mb. ruminantium. The results support the structure previously assigned to coenzyme F430.     

Spectroscopic and computational studies of reduction of the metal versus the tetrapyrrole ring of coenzyme F430 from methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the final step in methane biosynthesis by methanogenic archaea and contains a redox-active nickel tetrahydrocorphin, coenzyme F430, at its active site. Spectroscopic and computational methods have been used to study a novel form of the coenzyme, called F330, which is obtained by reducing F430 with sodium borohydride (NaBH4). F330 exhibits a prominent absorption peak at 330 nm, which is blue shifted by 100 nm relative to F430. Mass spectrometric studies demonstrate that the tetrapyrrole ring in F330 has undergone reduction, on the basis of the incorporation of protium (or deuterium), upon treatment of F430 with NaBH4 (or NaBD4). One- and two-dimensional NMR studies show that the site of reduction is the exocyclic ketone group of the tetrahydrocorphin. Resonance Raman studies indicate that elimination of this pi-bond increases the overall pi-bond order in the conjugative framework. X-ray absorption, magnetic circular dichroism, and computational results show that F330 contains low-spin Ni(II). Thus, conversion of F430 to F330 reduces the hydrocorphin ring but not the metal. Conversely, reduction of F430 with Ti(III) citrate to generate F380 (corresponding to the active MCR(red1) state) reduces the Ni(II) to Ni(I) but does not reduce the tetrapyrrole ring system, which is consistent with other studies [Piskorski, R., and Jaun, B. (2003) J. Am. Chem. Soc. 125, 13120-13125; Craft, J. L., et al. (2004) J. Biol. Inorg. Chem. 9, 77-89]. The distinct origins of the absorption band shifts associated with the formation of F330 and F380 are discussed within the framework of our computational results. These studies on the nature of the product(s) of reduction of F430 are of interest in the context of the mechanism of methane formation by MCR and in relation to the chemistry of hydroporphinoid systems in general. The spectroscopic and time-dependent DFT calculations add important insight into the electronic structure of the nickel hydrocorphinate in its Ni(II) and Ni(I) valence states.     

Resonance raman studies of a c type algal cytochrome. Deuterium shifts and a comparison with mammalian cytochrome c.

A c type cytochrome isolated from Synechococcus lividus grown on water and 2H2O media, has been studied by resonance Raman spectroscopy. The spectra were taken on the oxidized and reduced protein with excitation within the Soret band at 441.6 nm to determine whether individual resonance Raman bands of the heme shift upon deuterium substitution and also to provide a comparison with the spectra of horse heart cytochrome c. Some of the shifts observed with the deuterated heme c are larger than the corresponding shifts in meso-deuterated metalloporphyrins suggesting mixing of peripheral substituent vibrations with the skeletal modes of the porphyrin macrocycle. The algal cytochrome exhibits resonance Raman spectra roughly similar to those of horse heart cytochrome c, consistent with its optical absorption spectra which is typical of c type cytochromes, although a detailed comparison reveals note-worthy differences between the spectra of the two proteins; this may be a reflection of the effect of non-methionine ligands and protein environment on the vibrations of the c type heme in the algal cytochrome.     

Activation of heme-regulated eukaryotic initiation factor 2alpha kinase by nitric oxide is induced by the formation of a five-coordinate NO-heme complex: optical absorption, electron spin resonance, and resonance raman spectral studies

Heme-regulated eukaryotic initiation factor 2alpha kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the Fe(II)-HRI(NO) complex displayed 5-fold greater eukaryotic initiation factor 2alpha kinase activity than the Fe(III)-HRI complex. The Fe(III)-HRI complex exhibited a Soret peak at 418 nm and a rhombic ESR signal with g values of 2.49, 2.28, and 1.87, suggesting coordination with Cys as an axial ligand. Interestingly, optical absorption, ESR, and resonance Raman spectra of the Fe(II)-NO complex were characteristic of five-coordinate NO-heme. Spectral findings on the coordination structure of full-length HRI were distinct from those obtained for the isolated N-terminal heme-binding domain. Specifically, six-coordinate NO-Fe(II)-His was observed but not Cys-Fe(III) coordination. It is suggested that significant conformational change(s) in the protein induced by NO binding to the heme lead to HRI activation. We discuss the role of NO and heme in catalysis by HRI, focusing on heme-based sensor proteins.     

Ni(II) and Ni(I) forms of pentaalkylamide derivatives of cofactor F430 of Methanobacterium thermoautotrophicum.

A series of pentaalkylamide forms of F430 and of its 12,13-diepimer have been generated and characterized. Carbodiimide-assisted N-hydroxysulfosuccinimide activation of all five peripheral carboxylates of the F430 macrocycle allows nucleophilic attack by a number of primary amines (RNH2, R- = CH3-, CH3CH2-, CF3CH2-, CH3(CH2)3-) generating the pentaalkylamide derivatives. The identity of each derivative has been verified by fast-atom bombardment mass spectrometry (FAB-MS). The solubility of these derivatives in aprotic organic solvents varies as the amine alkyl substituent (R-) is changed. Electrochemical measurements have shown that the Ni(II/I) reduction potentials in N,N-dimethylformamide (DMF) are approximately -1 V (Ag/AgCl). Reduction by sodium amalgam in THF generates the Ni(I) form of the F430 diepimer pentabutylamide. The visible and EPR spectra of this Ni(I) species are very similar to the corresponding spectra of Ni(I) F430M (Jaun, B. and Pfaltz, A. (1986) J. Chem. Soc. Chem. Commun. 1327-1329.).     

Evidence for synergistic anion binding to iron in ovotransferrin complexes from resonance Raman and extended X-ray absorption fine structure analysis

The 2,3-dihydroxybenzoate and thioglycolate complexes of iron(III)-ovotransferrin have been studied with resonance Raman and extended x-ray absorption fine structure spectroscopies, respectively, to obtain evidence for the coordination of the synergistic anion to the iron center. The dihydroxybenzoate complex exhibits resonance-enhanced Raman vibrations arising from both the endogenous tyrosinates and the added dihydroxybenzoate. A comparison of the extended x-ray absorption fine structure spectra of the carbonate and thioglycolate complexes shows a large feature at about 1.95 A assigned to Fe-(O,N) interactions. The latter complex exhibits an added feature at 2.32 A assigned to an Fe-S interaction. These experiments demonstrate that the Lewis base functions in the synergistic anions coordinate to the iron in ovotransferrin.     

Empirical Force Field Analysis of the Revised Structure of Coenzyme F430. Epimerization and Geometry of the Corphinoid Tetrapyrrole

Abstract

We undertook an empirical force field analysis of the conformational changes that accompany the diepimeriztion of coenzyme F430. The crystal structure of 12,13-diepi F430M was used as a test of the parameter set and as the basis for the calculations. The individual pyrrole rings in 13-epi and 12,13-diepi F430 adopt alternating half chair conformations leading to a ruffled macrocycle, native F430 is also ruffled but the individual pyrroles are planar. The 12,13 di-dehydro F430 and native F430 conformations are extremely similar, this accounts for the experimental observation that reduction of 12,13 di-dehydro-F430 forms native F430 and not 12,13-diepi F430. Native F430 can easily accommodate both square planar and, by bending, trigonal bipyramidal coordination geometries about nickel. We suggest the bent trigonal bipyramidal form is the conformer bound to the protein and that direct binding of the amino acid side chains to nickel is probably not important.     

Denaturation and renaturation of myeloperoxidase. Consequences for the nature of the prosthetic group.

The effects of the chaotropic agent, guanidine HCl, on the chlorinating activity, optical absorption, EPR, and resonance Raman spectra of myeloperoxidase have been studied. In the presence of the agent the Soret optical absorption of the reduced enzyme (lambda max = 474 nm) is blue shifted to 448 nm, a position similar to heme alpha-containing enzymes. The chlorinating activity of the enzyme disappears, and EPR spectra show a loss of intensity of the rhombic high spin heme signals (gx = 6.9; gy = 5.4) and the appearance of a more axial high spin signal (gx = gy = 6.0). Surprisingly the effects of guanidine HCl are partly reversible. Upon decreasing the concentration of the chaotropic agents by dilution, both the chlorinating activity and the original optical spectrum of native reduced enzyme (lambda max = 474 nm) are partly restored. The resonance Raman spectra of denatured cyanomyeloperoxidase are less complicated than those of native myeloperoxidase, which have been interpreted previously to suggest an iron chlorin chromophore. The multiple lines in the oxidation state marker region are not seen in the spectra of the denatured species. The changes suggest that upon denaturation the macrocycle is converted into a more symmetric structure. Since the effects on the optical absorption spectrum are reversible we speculate that, in the native enzyme, an apparent porphyrin macrocycle undergoes a reversible interaction with amino acid residues in the protein which creates an asymmetry in the electronic distribution of the macrocycle. Comparison of the Raman spectra of denatured cyanomyeloperoxidase with those of analogous heme alpha model complexes suggests the presence of a formyl group in the denatured species; our data, however, demonstrate that the chromophore structure is not identical to heme alpha and may contain a different C beta substitution on the ring macrocycle.     

SOUL in mouse eyes is a new hexameric heme-binding protein with characteristic optical absorption, resonance Raman spectral, and heme-binding properties

SOUL is specifically expressed in the retina and pineal gland and displays more than 40% sequence homology with p22HBP, a heme protein ubiquitously expressed in numerous tissues. SOUL was purified as a dimer in the absence of heme from the Escherichia coli expression system but displayed a hexameric structure upon heme binding. Heme-bound SOUL displayed optical absorption and resonance Raman spectra typical of 6-coordinate low-spin heme protein, with one heme per monomeric unit for both the Fe(III) and Fe(II) complexes. Spectral data additionally suggest that one of the axial ligands of the Fe(III) heme complex is His. Mutation of His42 (the only His of SOUL) to Ala resulted in loss of heme binding, confirming that this residue is an axial ligand of SOUL. The K(d) value of heme for SOUL was estimated as 4.8 x 10(-9) M from the association and dissociation rate constants, suggesting high binding affinity. On the other hand, p22HBP was obtained as a monomer containing one heme per subunit, with a K(d) value of 2.1 x 10(-11) M. Spectra of heme-bound p22HBP were different from those of SOUL but similar to those of heme-bound bovine serum albumin in which heme bound to a hydrophobic cavity with no specific axial ligand coordination. Therefore, the heme-binding properties and coordination structure of SOUL are distinct from those of p22HBP, despite high sequence homology. The physiological role of the new heme-binding protein, SOUL, is further discussed in this report.     

Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes.

Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.     

Four- and five-coordinate species in nickel-reconstituted hemoglobin and myoglobin: Raman identification of the nickel-histidine stretching mode

Nickel(II)-reconstituted hemoglobin (NiHb) and myoglobin (NiMb) and model Ni porphyrins have been investigated by Soret-resonance Raman difference spectroscopy. Two sets of frequencies for the oxidation-state and core-size marker lines in the region from 1300 to 1700 cm-1 indicate two distinct sites in NiHb. Only one of these sites is evident in the Raman spectra of NiMb. This result is consistent with the UV-visible absorption spectrum of NiHb, which shows two Soret bands at 397 and 420 nm and one Soret at 424 nm for NiMb. Excitation at the blue Soret component of NiHb with 406.7-nm laser radiation preferentially enhances the set of Raman marker lines typical of Ni-protoporphyrin IX [Ni(ProtoP )] in noncoordinating solvents. The wavelength of the blue Soret component and the Raman spectrum indicate four-coordination for this site in NiHb. Laser excitation in the red Soret band enhances a set of lines whose frequencies are compatible with neither four- nor six-coordinate frequencies but are intermediate between the two. The red Soret band of the proteins is also considerably less red shifted than six-coordinate Ni-porphyrin models. These results suggest that Ni in the second site possesses a single axial ligand. Raman spectra of 64Ni-reconstituted and natural abundance Ni-reconstituted hemoglobins, obtained simultaneously in a Raman difference spectrometer, have identified the Ni-ligand stretch at 236 cm-1. The line shifts to 229 cm-1 for the 64Ni-reconstituted Hb. For a pure Ni-ligand stretch a 10-cm-1 shift would be predicted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elucidation of the differences between the 430- and 455-nm absorbing forms of P450-isocyanide adducts by resonance Raman spectroscopy.

Alkylisocyanide adducts of microsomal P450 exist in two interconvertible forms, each giving the Soret maximum around 430 or 455 nm. This is demonstrated with a rabbit liver P450 2B4. Resonance Raman spectra of the 430- and 455-nm forms were examined for typical P450s of the two types as well as for P450 2B4 because the 430-nm form of P450 2B4 is liable to change into P420. P450cam and P450nor were selected as a model of the 430- and 455-nm forms, respectively. For the n-butyl isocyanide (CNBu) adduct, the Fe(II)-CNBu stretching band was observed for the first time at 480/467 cm(-1) for P450cam and at 471/459 cm(-1) for P450nor with their (12)CNBu/(13)CNBu derivatives. For P450cam, but not P450nor, other (13)C isotope-sensitive bands were observed at 412/402, 844/835, and 940/926 cm(-1). The C-N stretching mode was identified by Fourier transform IR spectroscopy at 2116/2080 cm(-1) for P450cam and at 2148/2108 cm(-1) for P450nor for the (12)C/(13)C derivatives. These findings suggest that the binding geometry of isocyanide differs between the two forms-bent and linear structures for P450cam-CNBu and P450nor-CNBu, respectively. In contrast, in the ferric state, the Raman (13)C isotopic frequency shifts, and the IR C-N stretching frequencies (2213/2170 and 2215/2172 cm(-1)) were similar between P450cam and P450nor, suggesting similar bent structures for both.     

Resonance Raman spectra of horseradish peroxidase and bovine liver catalase compound I species. Evidence for predominant 2A2u pi-cation radical ground state configurations.

The nature of the porphyrin pi-cation radicals in the horseradish peroxidase and bovine liver catalase (BLC) compound I species have been investigated by studying their resonance Raman spectra. A variety of laser excitation and sample interrogation procedures have been employed in order to minimize previously documented problems arising from photoinduced conversions. With Soret band excitation, the spectra obtained for both species resemble that of a compound II-like photoproduct unless the samples are excited with residence times in the microsecond regime with very low (approximately 1 milliwatt) powers. When these precautions are taken, spectra attributable to the compound I species themselves are obtained. The spectrum for horseradish peroxidase compound I is similar to that reported by Paeng and Kincaid (Paeng, K.-J., and Kincaid, J. R. (1988) Am. Chem. Soc. 110, 7913-7915) using a similar approach. Both horseradish peroxidase and BLC compound I exhibit frequency shifts relative to their compound II species that are in the direction observed for model pi-cation radicals with predominant 2A2u character. The magnitudes of these shifts are smaller than those observed for heme models that lack aromatic axial ligands, but agree well with those observed on formation of the compound I analog of N alpha-acetyl microperoxidase-8 that has His as a proximal ligand. This observation is consistent with partial delocalization of the radical density onto the proximal His-170 and Tyr-357 ligands in horseradish peroxidase and BLC, respectively. The strong ligand field provided by these ligands on the proximal side and oxo ligand on the distal side of the heme group is apparently sufficient to reverse the 2A1u radical ground state preference observed for heme-like porphyrin species (e.g. octaethylporphyrins) with weak axial fields. Enhancement of several bands assigned to the Tyr-357 ligand has also been observed for BLC compound I with 406.7-nm excitation. This is attributed either to resonance with a tyrosinate----Fe(IV) charge transfer band or to the coupling provided by radical spin delocalization onto the tyrosinate ligand.     

Nickel requirement and factor F430 content of methanogenic bacteria.

Methanobacterium thermoautotrophicum has been reported to require nickel for growth and to contain high concentrations of a nickel tetrapyrrole designated factor F430. In this communication it is shown that all methanogenic bacteria investigated incorporated nickel during growth and also synthesized factor F430. This was also true for Methanobrevibacter smithii, which is dependent on acetate as a carbon source, and for Methanosarcina barkeri growing on acetate or methanol as energy sources. Other bacteria, including Acetobacterium woodii and Clostridium thermoaceticum, contained no factor F430. It is further shown that two yellow nickel-containing degradation products were formed from factor F430 when heated at pH 7. This finding explains why several forms of factor F430 were found in methanogenic bacteria when a heat step was employed in the purification procedure.