Chloroperoxidase: P-450 type absorption in the absence of sulfhydryl groups.

The oxidation state of the two half-cystine residues in the native ferric form of chloroperoxidase and in the reduced ferrous chloroperoxidase has been examined in order to evaluate the role of sulfhydryl groups as determinants of P-450 type spectra. M?ssbauer and optical spectroscopy studies indicate that the ferrous forms of P-450cam and chloroperoxidase have very similar or identical heme environments. Model studies have suggested that sulfhydryl groups may function as axial ligands for developing P-450 character. However, chemical studies involving both sulfhydryl reagents and amperometric titrations show that neither the ferric nor the chemically produced ferrous forms of chloroperoxidase contain a sulfhydryl group. These results rule out the hypothesis that sulfhydryl groups are unique components for P-450 absorption characteristics. The optical and electron paramagnetic resonance (EPR) spectra of the nitric oxide complex of chloroperoxidase have been obtained and compared to those of myoglobin, hemoglobin, and cytochrome c and horseradish peroxidase. The EPR spectrum of the NO-ferrous chloroperoxidase complex, which is similar to that of cytochrome P-450cam, does not show the extra nitrogen hyperfine structure which appears to be characteristic of those hemoproteins which have a nitrogen atom as an axial heme ligand.     

Model of the coordination site for cobalt-substituted cytochrome P450cam

Cobalt-substituted cytochrome P450_cam was recently reconstituted by Wagner et al (J. Biol. Chem. 256, 6266, (1981)). A model of its coordination site was constructed to determine the mode of axial coordination of the native enzyme. Complexes were prepared from cobalt porphyrins (cobalt-protoporphyrin IX (CoPPIX), cobalt-meso-tetraphenylporphyrin, cobalt-γ-laurylpyridyl triphenylporphyrin, and cobalt-octaethylporphyrin), thioglycolate ester, and tetramethylammonium hydroxide in organic solvents. Complexes prepared in an organic solvent such as CHC1₃ under air at room temperature exhibited a stable Soret hyperporphyrin spectrum characterized by split Soret bands, especially like that of the thiol-Co-P450_cam complex Comparison of the spectra of the hyperporphyrin spectral complexes titrated with various types of alcohol and imidazole, with the spectrum of Co-P450_cam in the oxidized state support the idea that an axial thiolate at the fifth position and a hydroxyl group of alcohol at the sixth position of the heme form the coordination site of Co-P450_cam The CoPPIX-thiolate-ethanol complex retaining S⁻-Co(III)-OH coordination is thought to be a possible model of Co-P450_cam in the oxidized state.     

Spin-equilibrium and heme-ligand alteration in a high-potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is +150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at congruent to 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 x 10(4) M-1 s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax congruent to 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.     

Resonance Raman investigations of chloroperoxidase, horseradish peroxidase, and cytochrome c using Soret band laser excitation.

Resonance Raman spectra of the heme protein chloroperoxidase in its native and reduced forms and complexed with various small ions are obtained by using laser excitation in the Soret region (350-450 nm). Additionally, Raman spectra of horseradish peroxidase, cytochrome P-450cam, and cytochrome c, taken with Soret excitation, are presented and discussed. The data support previous findings that indicate a strong analogy between the active site environments of chloroperoxidase and cytochrome P-450cam. The Raman spectra of native chloroperoxidase are found to be sensitive to temperature and imply that a high leads to low spin transition of the heme iron atom takes place as the temperature is lowered. Unusual peak positions are also found for native and reduced chloroperoxidase and indicate a weakening of porphyrin ring bond strengths due to the presence of a strongly electron-donating axial ligand. Enormous selective enhancements of vibrational modes at 1360 and 674 cm-1 are also observed in some low-spin ferrous forms of the enzyme. These vibrational frequencies are assigned to primary normal modes of expansion of the prophyrin macrocycle upon electronic excitation.     

1H nuclear magnetic resonance and magnetic circular dichroism studies of ferric low-spin cytochrome P-450scc

400 MHz ¹H NMR of ferric low-spin cytochrome P-450_scc purified from bovine adrenal cortex was measured for the first time. As compared with ¹H NMR spectra of low-spin P-450_cam and metMb- mercaptan complexes, paramagnetic shifts of low-spin P-450_scc complexes were more divergent, suggesting that there is a subtle difference in the heme environment between P-450_scc and P-450_cam [1]. The paramagnetic shifts of low-spin complexes of P-450_scc caused by adding nitrogenous inhibitors, aminoglutethimide and metyrapone, were different from those caused by adding an intermediate, 20α-hydroxycholesterol, and a detergent, Tween 20 [2]. The paramagnetic shifts of the metMb-mercaptan complexes were convergent compared with those of ferric low-spin P-450_scc and P-450_cam, suggesting that the electronic character and/or the conformation of the internal thiolate ligand in P-450_scc and P-450_cam are different from those of the external thiolate ligand in metMb-thiolate complexes [3]. The paramagetic shifts of the metMb-mercaptan complexes were dependent on the electron donating factor of the alkyl group of the bound mercaptans [4].Magnetic CD(MCD) spectra of ferric low-spin P-450_scc, rabbit liver P-450 complexes and metMb- mercaptan complexes were also observed at various temperatures. The temperature dependences of the Soret MCD bands for the low-spin P-450 and metMb- mercaptan complexes were decidedly less pronounced than those for the low-spin metMb-CN? or imidazole complexes, suggesting that thiolate ligands markedly influence the Soret MCD band of the ferric low-spin complexes [1]. The suggestion described in [2] implied by the ¹H NMR study was reconfirmed from the temperature dependence study of the Soret MCD [2]. The temperature dependences of the Soret MCD bands for low-spin P-450 complexes having a non-nitrogenous ligand were more pronounced than for those having a nitrogenous ligand.     

Absorption spectral studies on heme ligand interactions of P-450nor

Heme-external ligand interactions of P-450_nor were examined spectrophotometrically and compared with those of other P-450s. Most nitrogenous ligands induced type II spectral changes on binding to ferric P-450_nor, as did other P-450s. In contrast with other P-450s, 2-methylpyridine and 1-butanol induced type I changes in the spectrum of P-450_nor. No spectral interaction of ferrous P-450_nor with these ligands was observed. The absorption spectra of the alkyl isocyanide complexes of ferrous P-450_nor exhibited the Soret peak at 427 nm with a slight shoulder at around 455 nm at neutral pH, and this shoulder was intensified as the pH was increased, suggesting that the isocyanide complexes of P-450_nor existed in two states (the 430 and 455 nm states) which were in pH-dependent equilibrium in a similar manner to microsomal P-450s. However, the equilibrium was shifted mostly to the 430 nm state in the complexes of P-450_nor. The findings suggest that P-450_nor, especially its ferrous form, has some distinct features from P-450_cam and microsomal P-450s in the distal heme environment.     

Efficient catalytic turnover of cytochrome P450(cam) is supported by a T252N mutation

A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450_cin (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [D.B. Hawkes, G.W. Adams, A.L. Burlingame, P.R. Ortiz de Montellano, J.J. De Voss, J. Biol. Chem. 277 (2002) 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450_cam (CYP101) was mutated to generate the T252N, T252N/V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher K_m values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher K_m values reflect a decrease in the camphor binding affinity. Non-productive H₂O₂ generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450_cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H₂O₂ and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.     

Oxidation-reduction potential measurements on chloroperoxidase and its complexes.

The oxidation-reduction potential of chloroperoxidase, an enzyme which catalyzes peroxidative chlorination, bromination, and iodination reactions, has been investigated. In addition to catalyzing biological halogenation reactions, chloroperoxidase is unusual in that the carbon monoxide complex of ferrous chloroperoxidase shows the typical long wavelength Soret absorption associated with P-450 hemoproteins. The pH dependence of the chloroperoxidase oxidation-reduction potential shows a discontinuity around pH 4.7. Similarly, measurements of the affinity of ferrous chloroperoxidase for carbon monoxide monitored both by spectroscopic and potentiometric titration exhibit a discontinuity in the pH 4.7 region. Oxidation-reduction potential measurements on chloroperoxidase in a CO atmosphere also show a discontinuous pH profile. These results suggest that ferrous chloroperoxidase undergoes reversible modification at low pH and that these changes are reflected in the oxidation-reduction potential. The oxidation-reduction potential of chloroperoxidase at pH 6.9 is - 140 mV, close to that measured for cytochrome P-450cam in the presence of substrate. The oxidation-reduction potential of chloroperoxidase at pH 2.7, the pH optimum for enzymatic chlorination, is +150 mV. The oxidation-reduction potentials of the halide complexes of chloroperoxidase (chloride, bromide, and iodide) are essentially identical with the potential measurements on the native enzyme. These observations suggest that, although halide anions bind to the enzyme, they probably do not bind as an axial ligand to the heme ferric iron.     

NMR studies of P-450 model systems: new structural probes for sulfur-containing hemoproteins.

A simple model for ferrous cytochrome P-450 has been investigated by proton and carbon-13 Fourier transform NMR. In the proton spectrum of the β-phenethyl mercaptan-protoheme-CO complex, the protons α and β to mercaptide sulfur are observed 2.62 and 0.62 ppm upfield of tetramethylsilane. The ¹³CO spectra show characteristic shifts at 204.7 and 197.0 δ for neutral and deprotonated mercaptan complexes, respectively.     

Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo-halide ion binding to chloroperoxidase

The proton nuclear magnetic resonance spectra of several chloroperoxidase-inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide-chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin.     

Preparation and properties of ferrous chloroperoxidase complexes with dioxygen, nitric oxide, and an alkyl isocyanide. Spectroscopic dissimilarities between the oxygenated forms of chloroperoxidase and cytochrome P-450

Extensive spectroscopic investigations of chloroperoxidase and cytochrome P-450 have consistently revealed close similarities between these two functionally distinct enzymes. Although the CO-bound ferrous states were the first to display such resemblance, additional comparisons have focused on the native ferric and ferrous and the ligand-bound ferric derivatives of the enzymes. In order to test the extent to which the spectral properties of the two enzymes match each other, we have prepared the NO, alkyl isocyanide, and O2 adducts of ferrous chloroperoxidase, the latter two for the first time. As expected, the NO adducts of the two proteins have similar UV-visible absorption and magnetic circular dichroism spectra; the same behavior is observed for the alkyl isocyanide complexes. Unexpectedly, the dioxygen adduct of ferrous chloroperoxidase (i.e. Compound III), generated in cryogenic solvents at -30 degrees C by bubbling with O2, is spectrally distinct from oxy-P-450-CAM. Identification of this derivative as oxygenated chloroperoxidase is based on the following criteria: It is EPR-silent at 77 K. The bound O2 is dissociable as judged by the uniform conversion to the CO-bound form. Oxy-chloroperoxidase autoxidizes to form the native ferric enzyme without detectable intermediates at a rate comparable to that determined for oxy-P-450-CAM. Oxy-chloroperoxidase exhibits optical absorption (lambda nm (epsilon mM) = 354 (41), 430 (94), 554 (16.5), 587 (12.5)) and magnetic circular dichroism spectra that are clearly distinct from those of histidine-ligated heme proteins such as oxy-myoglobin or oxy-horseradish peroxidase. Surprisingly, several of its spectral properties, namely the red-shifted Soret peak and discrete alpha peak, are also unlike those of oxy-P-450-CAM. Since considerable evidence has accumulated supporting the ligation of an endogenous thiolate to the heme iron of chloroperoxidase, as has been established for the P-450 enzyme, the observed dissimilarities suggest that the electronic properties of the two dioxygen adducts are quite sensitive to differences in their active site heme environment. This, in turn may be related to the functional differences between the two enzymes.     

Studies of the unique nature of the cytochrome P-450 active site. Electron spin resonance spectroscopy as a probe of the hemin iron of cytochrome P-450: the influence of buffer composition, alcohols, and nitrogenous ligands.

The electron spin resonance (esr) spectra of the low-spin form of hepatic microsomal cytochrome P-450 and of cytochrome P-450 isolated from Pseudomonas putida grown on d-camphor (P-450_cam) were studied in order to gain an understanding of the sensitivity of the hemin iron to changes in buffer. The shapes of the g_x and g_y esr signals of both the membrane-bound microsomal and soluble bacterial cytochromes P-450 were dependent upon buffer composition. With either system, the g_x and g_y signals were symmetric in some buffers and asymmetric in others. However, in potassium phosphate buffer, the esr spectra of low-spin cytochrome P-450 in microsomes isolated from phenobarbital (PB)- or 3-methylcholanthrene (3-MC-induced rats and cytochrome P-450_cam are similar with symmetric g_x and g_y signals. The esr spectrum of the low-spin form of cytochrome P-450 in isolated hepatocytes is similar to that of the microsomal and bacterial enzyme, again with a symmetric g_x signal. The effects of alcohols and nitrogenous ligands on the esr spectrum of the low-spin form were also investigated. The data indicate that extreme care must be exercised when interpreting esr spectra with respect to possible cytochrome P-450 heterogeneity in the microsomal membrane. The conditions for studying substrate interactions with microsomal cytochrome P-450 must also take into account these changes in symmetry of the esr spectrum.     

The pentaammineruthenium(III)histidine complex in ribonuclease A: application to the assignment of histidine proton resonances

The assignment of two histidine proton resonances in the proton NMR spectrum of ribonuclease A has been made by forming a paramagnetic complex between pentaammineruthenium(III) and the N-3 nitrogen of a single histidine residue. Reaction of chloropentaammineruthenium(III)dichloride with ribonuclease A in 0.1 m Tris-HCl, pH 7.0, 25°C yields a variety of products in which various histidine residues have been labeled. Cation-exchange chromatography affords the isolation of a specific derivative, labeled at a single histidine residue, that retains 66% of the activity toward the hydrolysis of 2′,3′-cyclic CMP. The site of labeling was determined by peptide mapping to be histidine 105. The binding of ruthenium results in the disappearance of both a histidine C-2 and a C-4 proton resonance from the downfield region of the proton NMR spectrum, as expected from model compound studies. The assignment of these two resonances to histidine 105 is in agreement with a previous assignment (J. L. Markley, 1975, Biochemistry, 14, 3546–3554), thereby demonstrating the potential utility of this ruthenium reagent in the assignment of histidine resonances in the proton NMR spectra of other proteins.     

Superoxide anion production by the autoxidation of cytochrome P450cam

Chemiluminescence occurs on autoxidation of oxygenated ferrous cytochrome P450_cam and is abolished by reagents that scavenge free radicals, by superoxide dismutase and singlet oxygen quenchers. We attribute the chemiluminescence to the decay of an excited singlet oxygen which arises from a superoxide anion radical precursor.     

Chemotaxis by Pseudomonas putida (ATCC 17453) towards camphor involves cytochrome P450cam (CYP101A1)

The camphor-degrading microorganism, Pseudomonas putida strain ATCC 17453, is an aerobic, gram-negative soil bacterium that uses camphor as its sole carbon and energy source. The genes responsible for the catabolic degradation of camphor are encoded on the extra-chromosomal CAM plasmid. A monooxygenase, cytochrome P450_cam, mediates hydroxylation of camphor to 5-exo-hydroxycamphor as the first and committed step in the camphor degradation pathway, requiring a dioxygen molecule (O₂) from air. Under low O₂ levels, P450_cam catalyzes the production of borneol via an unusual reduction reaction. We have previously shown that borneol downregulates the expression of P450_cam. To understand the function of P450_cam and the consequences of down-regulation by borneol under low O₂ conditions, we have studied chemotaxis of camphor induced and non-induced P. putida strain ATCC 17453. We have tested camphor, borneol, oxidized camphor metabolites and known bacterial attractants (d)-glucose, (d) - and (l)-glutamic acid for their elicitation chemotactic behavior. In addition, we have used 1-phenylimidazole, a P450_cam inhibitor, to investigate if P450_cam plays a role in the chemotactic ability of P. putida in the presence of camphor. We found that camphor, a chemoattractant, became toxic and chemorepellent when P450_cam was inhibited. We have also evaluated the effect of borneol on chemotaxis and found that the bacteria chemotaxed away from camphor in the presence of borneol. This is the first report of the chemotactic behaviour of P. putida ATCC 17453 and the essential role of P450_cam in this process.     

The cytochromes P450 and b5 and their reductases—Promising targets for structural studies by advanced solid-state NMR spectroscopy

Members of the cytochrome P450 (cyt P450) superfamily of enzymes oxidize a wide array of endogenous and xenobiotic substances to prepare them for excretion. Most of the drugs in use today are metabolized in part by a small set of human cyt P450 isozymes. Consequently, cyt P450s have for a long time received a lot of attention in biochemical and pharmacological research. Cytochrome P450 receives electrons from cytochrome P450 reductase and in selected cases from cytochrome b₅ (cyt b₅). Numerous structural studies of cyt P450s, cyt b₅, and their reductases have given considerable insight into fundamental structure-function relationships. However, structural studies so far have had to rely on truncated variants of the enzymes to make conventional X-ray crystallographic and solution-state NMR techniques applicable. In spite of significant efforts it has not yet been possible to crystallize any of these proteins in their full-length membrane bound forms. The truncated parts of the enzymes are assumed to be α-helical membrane anchors that are essential for some key properties of cyt P450s. In the present contribution we set out with a basic overview on the current status of functional and structural studies. Our main aim is to demonstrate how advanced modern solid-state NMR spectroscopic techniques will be able to make substantial progress in cyt P450 research. Solid-state NMR spectroscopy has sufficiently matured over the last decade to be fully applicable to any membrane protein system. Recent years have seen a remarkable increase in studies on membrane protein structure using a host of solid-state NMR techniques. Solid-state NMR is the only technique available today for structural studies on full-length cyt P450 and full-length cyt b₅. We aim to give a detailed account of modern techniques as applicable to cyt P450 and cyt b₅, to show what has already been possible and what seems to be viable in the very near future.     

Connectivity of proton and carbon spectra of the blue copper protein,plastocyanin, established by two-dimensional nuclear magnetic resonance

NMR studies of plastocyanin have centered on the ligands to the copper atom at the active site, particularly histidines-37 and -87. Heteronuclear (¹³C, ¹H) J-connectivity spectroscopy has enabled cross assignment of ¹H and ¹³C NMR resonances from the two copper-ligated histidines. In addition to providing assignments of the ¹³C resonances, the two-dimensional Fourier transform NMR results require the reversal of the original ¹H NMR assignments to the ring protons of histidine-37. The line widths of the ring protons of histidine-87 are field-dependent leading to determination of the reduced lifetime of the proton on the N_δ atom (about 400 μs).     

Cytochrome P450 cam: SS- dimer and -SH derivative reactivities.

N-ethyl-maleimide alkylation converts ferric P450_cam to a (succinimido-cys)₄ protein with native optical and EPR spectra but insensitive to substrate induced shift of iron to high spin with Soret absorption to higher energy and inactive in putidaredoxin ferric-ferrous reduction. On photo or chemical reduction the ferrous protein oxygenates and, with reduced putidaredoxin, converts substrate to product. Mild oxidation of P450_cam yields a disulfide dimer whose properties on alkylation of 3 sulfhydryls equal the succinimido₄ monomer; additional alkylation converts either monomer or dimer to a P420.     

Thermodynamic aspects of the CO-binding reaction to cytochrome P-450cam. Relevance with their biological significance and structure

The CO-binding kinetics of cytochrome P-450_cam(+) and P-450cam(−) have been measured in the millisecond time domain using a flash photolysis method. We have determined the reaction coordinates for free energy, enthalpy and entropy from the temperature dependence of the overall rate constants of the bimolecular forward (on) and backward (off) reactions. Comparing the thermodynamic profiles of P-450_cam with that of myoglobin (Mb) reported so far, the enthalpy and the entropy coordinates exhibit the following remarkable characteristics. The CO-binding equilibrium: The stability of the CO-complex is perfectly entropy-driven for P-450_cam, while enthalpy-driven for Mb. This entropy-driven feature for P-450_cam is enhanced by the dissociating d-camphor. The on and off activation processes: The on and off reactions for P-450_cam.are dominantly controlled by the enthalpy and entropy terms, respectively, while those for Mb are rather the reverse of the case of P-450_cam. The dissociation of d-camphor has a significant effect on the on reaction but no effect on the off reaction. Analyzing these thermodynamic features on the basis of the physical chemistry in the solution reaction, it was found that these characteristic profiles arise from the difference in the global structural change between the proteins. Namely, during the equilibrium process of the CO binding, this structural change is accompanied by a larger increase in the degree of freedom in P-450_cam than in Mb. We discussed the correlations between the structural changes and their biological significance.     

Protonation and dimerization reactions of cyclopentadienylrhodiumcarbonylphosphine and phosphite derivatives

Eight cyclopentadienylrhodiumcarbonylphosphine and phosphite complexes have been prepared and their IR, ¹H, and ³¹P NMR spectra recorded. A good correlation between carbonyl stretching frequencies and rhodium-phosphorous coupling constants has been observed. Reaction of these compounds with trifluorosulfonic acid, HCF₃SO₃, forms the expected cationic rhodium-hydride species which were examined using ¹H and ³¹P NMR spectroscopy. Similar reactions between trifluoroacetic acid, HCF₃CO₂, and the phosphine compounds gave evidence of rapid proton exchange at the metal. Reaction between trifluoroacetic acid and cyclopentadienylrhodiumcarbonylphosphite compounds yielded new sets of rhodium-hydride resonances which were shown to be due to the formation of dinuclear rhodium complexes and cyclopentadienylrhodiumbis(phosphite) complexes which arise under the reaction conditions. A Scheme for the formation of these reaction products is presented which is consistent with all of the experimental data.