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.     

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.     

Apoprotein formation and heme reconstitution of cytochrome P-450cam

Apoprotein suitable for heme reconstitution has been prepared by an acid/butanone extraction of cytochrome P-450cam at pH 2.5. Absorption spectra of apo-P-450cam indicate less than 2% residual holoenzyme. Four tryptophan residues per molecule were estimated from the aromatic absorbance region of denatured apoprotein. Heme-reconstituted holoprotein was purified in 30% yield to a specific activity equivalent to the native enzyme. Absorption and EPR spectra of 57Fe- and 54Fe-heme-enriched P-450cam reveal complete restoration of the native active site.     

Resonance Raman investigation of a soluble cytochrome c552 from alkaliphilic Bacillus firmus RAB

The environment of the heme site of a low-potential soluble cytochrome (c552) from alkaliphilic Bacillus firmus RAB has been characterized with resonance Raman scattering and compared to that of horse heart cytochrome c. The Raman data indicate that vibrational bands sensitive to the axial ligation of the heme, as well as modes sensitive to the heme peripheral environment in cytochrome c552, are distinct from those of horse heart cytochrome c. The spectra of cytochrome c552 display resonance Raman modes indicative of a methionine as the sixth ligand in the oxidized form, while the reduced form appears to contain a nitrogenous-based sixth ligand. In addition, Q-band excitation reveals differences among vibrational modes in cytochrome c552 that are sensitive to the amino acid environment surrounding the heme.     

Circular dichroism studies of low-spin ferric cytochrome P-450CAM ligand complexes

Circular dichroism (CD) spectroscopy has been used to probe the active site of bacterial ferric cytochrome P-450CAM. The endogenous sixth ligand to the heme iron has been displaced by an extensive series of exogenous oxygen, nitrogen, sulfur and other neutral and anionic donor ligands in an attempt to examine systematically the steric and electronic factors that influence the coupling of the heme chromophore to its protein environment. General trends for each ligand class are reported and discussed. Both the wavelengths and the intensities of the CD bands vary with ligand type and structure. All but one of the complexes exhibit negative CD maxima in their delta and Soret bands. Comparison to ferric myoglobin-thiolate complexes indicates that the negative sign observed for the cytochrome P-450 spectra is not a property of the thiolate fifth ligand, but rather arises from a different interaction of the cytochrome P-450 heme with its protein environment. Complexes with neutral oxygen donors display CD spectra that most closely resemble the spectrum of the native low-spin enzyme. Hyperporphyrin (split Soret) cytochrome P-450 complexes with thiolates, phosphines and cyanide trans to cysteinate have complex CD spectra, reflecting the intrinsic non-degeneracy of the Soret pi pi transitions. The extensive work presented herein provides an empirical foundation for use in analyzing the interaction of heme chromophores with their protein surroundings, not only for the cytochrome P-450 monooxygenases but also for heme proteins in general.     

An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.

Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5.     

Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.     

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.     

Electron-conformational interactions at the active site of reduced bacterial cytochrome P450cam induced by a substrate and analysis of the electron structure of heme]

Magnetic circular dichroism (MCD) spectra in the Soret region (360-480 nm) of camphor-free and camphor-bound reduced bacterial cytochrome P450cam from Pseudomonas putida were recorded and analysed in the temperature range from 2 K to 290 K. The temperature dependences of the MCD intensity are qualitatively changed by binding of substrate to the enzyme. In the absence of camphor the linear increase of the MCD intensity with 1/T at T < 4.2 K gives evidence for degeneracy or near degeneracy of the ground electronic state. In the presence of substrate the degeneracy is removed and temperature profiles show saturation behaviour at T < 4.2 K and wavelength dependence of their high-temperature parts. The temperature profiles for the long-wavelength region of the Soret band have a maximum approximately at 15 K, whereas the MCD intensity increases in a monotonous manner up to saturation in the short-wavelength region. The wavelength dependence of temperature profiles gives evidence for the co-existence of two different forms of substrate-bound reduced P450cam. The following conclusions were obtained from a theoretical analysis of the temperature profiles. In the absence of substrate there are very small if any rhombic distortions at the heme iron, and a parameter D of axial zero-field splitting is negative (D = -8.3 cm-1 and -6.2 cm-1 for P450cam and P450LM2, respectively). In the presence of substrate the two forms of reduced P450cam have positive parameters D but of different values (D1 = 12 cm-1 and D2 = 28 cm-1), and there are large rhombic distortions at the heme iron. More than two-fold difference between the D values made it possible to isolate temperature-dependent contributions of the two enzyme forms from the total MCD spectra and to simulate the alterations of the MCD spectra with temperature for reduced P450cam in the presence of substrate. Taking into account the drastic effect of substrate binding on the ground electronic state of reduced P450cam one can suggest that substrate binding induces the transition of enzyme from an inactive to an active state.     

Cobalt-substituted cytochrome P-450cam

Reconstitution of the apo-cytochrome with cobalt protoporphyrin provides a faithful P-450cam analogue as characterized by optical, ligand-binding, and enzymatic parameters. The thiol and cyanide complexes exhibit Soret "hyper" spectra, not previously observed in cobalt porphyrins. Substrate-induced spectral changes and limited stereospecific hydroxylation activity are retained in the cobalt P-450cam. The EPR (electron paramagnetic resonance) spectra of the reduced cobaltous protein indicate clearly an endogenous axial ligand other than a nitrogenous base and support an assignment of thiolate coordination. A thiolate ligand is also indicated by EPR measurements in the oxygenated cobaltous analogue. By analogy, these studies suggest that the native ferrous and oxygenated P-450cam states retain a thiolate axial ligand.     

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.     

Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2

Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.     

Magnetic circular dichroism studies of hepatic microsomal cytochrome P-450.

Magnetic circular dichroism (MCD) spectra have been measured for cytochrome P-450 (P-450) purified from phenobarbital-induced rabbit liver microsomes. The temperature dependence of some of the MCD spectra has also been determined. The MCD spectrum of oxidized P-450 seems to suggest that it is in a state intermediate between the ferric low-spin states. Model experiments suggest that this anomaly arises from the coordination of a thiolate anion to the heme. Reduced P-450 shows a very peculiar MCD spectrum; the spectrum as well as its temperature dependence suggest that the heme in reduced P-450 is a "mixture" in terms of redox and/or spin states. The MCD spectrum of the CO complex of reduced P-450 exhibits an apparent Faraday A term around 450 nm which consists of about 50% C term and 50% the other terms, indicating that it is not in a purely ferrous low-spin state. The CO complex of reduced cytochrome P-420 (P-420), on the other hand, shows an MCD spectrum characteristic of a ferrous low-spin heme. It is suggested from model experiments that the thiolate anion coordinates to the heme trans to CO in the P-450-CO complex. The Soret region of the MCD spectrum of the EtNC complex of reduced P-450 is characterized by two apparent A terms around 430 and 455 nm, whereas that of the corresponding complex of P-420 has only one apparent A term around 434 nm.     

M?ssbauer investigations of high-spin ferrous heme proteins. II. Chloroperoxidase, horseradish peroxidase, and hemoglobin.

Reduced samples of chloroperoxidase, horseradish peroxidase, and deoxyhemoglobin were studied by M?ssbauer spectroscopy in strong magnetic fields. The intricate paramagnetic spectra of chloroperoxidase were evaluated in detail in the framework of a spin Hamiltonian pertinent to high-spin ferrous iron. The studies strongly suggest that, in their reduced states, chloroperoxidase from Caldariomyces fumago and cytochrome P-450 from Pseudomonas putida have similar, if not identical ligand structures of the heme iron. The spectral similarities of these two proteins, noted in an earlier M?ssbauer investigation, are further explored and substantiated. Reduced horseradish peroxidase and deoxyhemoglobin, on the other hand, show high-field M?ssbauer spectra that differ considerably from each other and, in particular, from those of the P-450 type, suggesting a different ligand arrangement of the heme iron for each case.     

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.     

Complexes of trivalent oxygenated phosphorus compounds with cytochrome P-450 and cytochrome P-420: the origin of double Soret spectra.

Trivalent oxygenated phosphorus ligands include alkyl and aryl phosphites, (RO)3P, phosphonites, (RO)2PR, and phosphinites, ROPR2. All such compounds tested, with the exception of triphenyl phosphite, interact with ferrous cytochrome P-450 and its denatured form, cytochrome P-420, to produce complexes having two peaks in the Soret region of their optical difference spectra. Careful evaluation of these spectra indicate that they arise for different reasons for each of the two cytochromes. Clear evidence shows that cytochrome P-450 is not denatured by these ligands. The high affinity of these ligands for heme iron is indicated by small Ks values. The experimental results are used to substantiate a theory of the origin of microsomal double Soret spectra and the nature of the environments available for microsomal cytochromes P-450 and P-420.     

Resonance Raman detection of the Fe-S bond in endothelial nitric oxide synthase

We report the first low-frequency resonance Raman spectra of ferric endothelial nitric oxide synthase (eNOS) holoenzyme, including the frequency of the Fe-S vibration in the presence of the substrate L-arginine. This is the first direct measurement of the strength of the Fe-S bond in NOS. The Fe-S vibration is observed at 338 cm(-1) with excitation at 363.8 nm. The assignment of this band to the Fe-S stretching vibration was confirmed by the observation of isotopic shifts in eNOS reconstituted with 54Fe- and 57Fe-labeled hemin. Furthermore, the frequency of this vibration is close to those observed in cytochrome P450(cam) and chloroperoxidase (CPO). The frequency of this vibration is lower in eNOS than in P450(cam) and CPO, which can be explained by differences in hydrogen bonding to the proximal cysteine heme ligand. On addition of substrate to eNOS, we also observe several low-frequency vibrations, which are associated with the heme pyrrole groups. The enhancement of these vibrations suggests that substrate binding results in protein-mediated changes of the heme geometry, which may provide the protein with an additional tuning element for the redox potential of the heme iron. The implications of our findings for the function of eNOS will be discussed by comparison with P450(cam) and model compounds.     

Resonance Raman spectral isolation of the a and a3 chromophores in cytochrome oxidase.

Resonance Raman spectra of reduced CO-bound cytochrome oxidase obtained at two different excitation frequencies (441.6 and 413.1 nm) are compared with the spectra of the fully reduced enzyme. In the spectra of the CO-bound complex only the cytochrome a modes are strongly enhanced with 441.6 nm excitation and only the modes of the CO-bound cytochrome a3 heme are strongly enhanced with 413.1-nm excitation. In the fully reduced complex with both excitation frequencies, modes of both cytochrome a and a3 are enhanced. By subtraction we are able to uncover the complete spectrum of the fully reduced ligand-free cytochrome a3 heme. Thus, we report the discrete resonance Raman spectra of cytochromes a2+, a2+3, and a2+3 (CO). The spectra of fully reduced cytochrome a and ligand-free cytochrome a3 are very different especially in the low frequency region. Binding CO to ferrous cytochrome a3 results in electronic structure changes in the heme analogous to those in hemoglobin and myoglobin, from which we conclude that there is nothing electronically unique in the ferrous cytochrome a3 heme to account for its catalytic properties.     

Effects of cholesterol and adrenodoxin binding on the heme moiety of cytochrome P-450scc: a resonance Raman study

The effects of cholesterol and adrenodoxin binding on resonance Raman spectra of cytochrome P-450scc in both oxidized and CO-reduced states were examined. Upon cholesterol binding, oxidized cytochrome P-450scc showed a significant shift of spin equilibrium from low-spin to high-spin state. Addition of adrenodoxin caused a complete conversion of cholesterol-bound oxidized cytochrome P-450scc to a pure high-spin state that was considered to be in the hexacoordinated state judged by the v10 mode at 1620 cm-1 and v3 mode around 1485 cm-1. Cholesterol in substrate binding site may oppose a linear and perpendicular binding of carbon monoxide to the reduced heme iron, leading to the distorted Fe-C-O linkage. This is based on the following observations: (1) an increase of the Fe-CO stretching frequency to 483 from 477 cm-1 upon addition of cholesterol; (2) an enhanced photodissociability of bound carbon monoxide of CO complex of cytochrome P-450scc in the presence of cholesterol. As another aspect of the effect of cholesterol on the CO complex form of cytochrome P-450scc, the enhanced stability of the native form ("P-450" form) was observed. There was no additional effect of reduced adrenodoxin on the Raman spectra of the CO-reduced form of cytochrome P-450scc.     

Resonance Raman detection of bound dioxygen in cytochrome P-450cam

We have used resonance Raman spectroscopy and isotopic labeling techniques to unambiguously assign the dioxygen stretching frequency (vo-o) in the substrate-bound oxygenated complex of cytochrome P-450cam. The frequency found for Vo-o in the P-450cam system (1140 cm-1) is in remarkable agreement with recent studies of thiolate heme model compounds. The general features of the oxy-P-450cam Raman spectra are tabulated and comparisons are made with the oxy complexes of hemoglobin, myoglobin, and various model compounds. Most of the results are qualitatively explained by consideration of electron donation into the pi g (O2)/d pi (M) orbitals of the oxygenated complex (M = Fe or Co). It is also noted that the effect of the "extra" electron in the nitrogen base Co(II) oxy complexes, in some ways, parallels the effect of the lone pair electrons of thiolate in the oxy-P-450cam complex. This is evidenced by the enhanced resonance Raman activity of vo-o in both the Co(II) and P-450 systems as well as by the similarity of the vo-o frequencies.