Resilience of Rhodobacter sphaeroides cytochrome bc1 to heme c1 ligation changes

Typically, c hemes are bound to the protein through two thioether bonds to cysteines and two axial ligands to the heme iron. In high-potential class I c-type cytochromes, these axial ligands are commonly His-Met. A change in this methionine axial ligand is often correlated with a dramatic drop in the heme redox potential and loss of function. Here we describe a bacterial cytochrome c with an unusual tolerance to the alternations in the heme ligation pattern. Substitution of the heme ligating methionine (M185) in cytochrome c1 of the Rhodobacter sphaeroides cytochrome bc1 complex with Lys and Leu lowers the redox midpoint potential but not enough to prevent physiologically competent electron transfer in these fully functional variants. Only when Met-185 is replaced with His is the drop in the redox potential sufficiently large to cause cytochrome bc1 electron transfer chain failure. Functional mutants preserve the structural integrity of the heme crevice: only the nonfunctional His variant allows carbon monoxide to bind to reduced heme, indicating a significant opening of the heme environment. This range of cytochrome c1 ligand mutants exposes both the relative resilience to sixth axial ligand change and the ultimate thermodynamic limits of operation of the cofactor chains in cytochrome bc1.     

Crystal structure of Nitrosomonas europaea cytochrome c peroxidase and the structural basis for ligand switching in bacterial di-heme peroxidases.

The crystal structure of the fully oxidized di-heme peroxidase from Nitrosomonas europaea has been solved to a resolution of 1.80 A and compared to the closely related enzyme from Pseudomonas aeruginosa. Both enzymes catalyze the peroxide-dependent oxidation of a protein electron donor such as cytochrome c. Electrons enter the enzyme through the high-potential heme followed by electron transfer to the low-potential heme, the site of peroxide activation. Both enzymes form homodimers, each of which folds into two distinct heme domains. Each heme is held in place by thioether bonds between the heme vinyl groups and Cys residues. The high-potential heme in both enzymes has Met and His as axial heme ligands. In the Pseudomonas enzyme, the low-potential heme has two His residues as axial heme ligands [Fulop et al. (1995) Structure 3, 1225-1233]. Since the site of reaction with peroxide is the low-potential heme, then one His ligand must first dissociate. In sharp contrast, the low-potential heme in the Nitrosomonas enzyme already is in the "activated" state with only one His ligand and an open distal axial ligation position available for reaction with peroxide. A comparison between the two enzymes illustrates the range of conformational changes required to activate the Pseudomonas enzyme. This change involves a large motion of a loop containing the dissociable His ligand from the heme pocket to the molecular surface where it forms part of the dimer interface. Since the Nitrosomonas enzyme is in the active state, the structure provides some insights on residues involved in peroxide activation. Most importantly, a Glu residue situated near the peroxide binding site could possibly serve as an acid-base catalytic group required for cleavage of the peroxide O--O bond.     

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.     

Some properties of mammalian cardiac cytochrome c1.

Investigations into the nature of the axial heme ligands, the strength of the heme crevice, the reactivity with cyanide, and the ascorbate reducibility of cytochrome c1 were performed to explore structure-function relationships of cytochrome c1. The existence of an absorbance band at 690 nm, which was quenched by raising the pH with a pK of 9.2 corresponding to a low spin-low transition, suggested that a methionine residue probably functioned as one of the axial heme iron ligands in this cytochrome. Spectral titrations of cytochrome c1 in the low pH range showed a markedly elevated pK for the low spin-high spin transition relative to cytochrome c. Denaturation studies with urea, the absence of any reaction with cyanide, and the evidence from other lines would appear to indicate that the heme group of cytochrome c1 was reduced by ascorbate at approximately 5% of the rate of reduction of cytochrome c but this rate dramatically increased with increasing pH concomitant with the disappearance of the 690 nm absorbance band. Circular dichroic spectra substantiated that elevated pH produced conformational changes localized to the heme crevice and probably also the regions containing aromatic residues. The enhanced rate of ascorbate reduction was perhaps a consequence of the increased accessibility of the heme iron to ascorbate. Major unfolding of the protein in 8 M urea, however, completely abolished the ascorbate reducibility of cytochrome c1. The buried nature of the heme group of cytochrome c1 would probably preclude transfer of an electron from cytochrome c1 to cytochrome c through a direct Fe-Fe or a heme-heme interaction. This poses an important question concerning the mechanism of this electron transfer between these two cytochromes not only in mitochondria but also in solution.     

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.     

The axial ligands of heme in cytochromes: a near-infrared magnetic circular dichroism study of yeast cytochromes c, c1, and b and spinach cytochrome f

Room temperature near-infrared magnetic circular dichroism and low-temperature electron paramagnetic resonance measurements have been used to characterize the ligands of the heme iron in mitochondrial cytochromes c, c1, and b and in cytochrome f of the photosynthetic electron transport chain. The MCD data show that methionine is the sixth ligand of the heme of oxidized yeast cytochrome c1; the identify of this residue is inferred to be the single conserved methionine identified from a partial alignment of the available cytochrome c1 amino acid sequences. A different residue, which is most likely lysine, is the sixth heme ligand in oxidized spinach cytochrome f. The data for oxidized yeast cytochrome b are consistent with bis-histidine coordination of both hemes although the possibility that one of the hemes is ligated by histidine and lysine cannot be rigorously excluded. The neutral and alkaline forms of oxidized yeast cytochrome c have spectroscopic properties very similar to those of the horse heart proteins, and thus, by analogy, the sixth ligands are methionine and lysine, respectively.     

Cytochromes: Reactivity of the "dark side" of the heme

Ligand binding to the heme distal side is a paradigm of heme-protein biochemistry, the proximal axial ligand being in most cases a His residue. NO binds to the ferrous heme-Fe-atom giving rise to hexa-coordinated adducts (as in myoglobin and hemoglobin) with His and NO as proximal and distal axial ligands, respectively, or to penta-coordinated adducts (as in soluble guanylate cyclase) with NO as the axial distal ligand. Recently, the ferrous derivative of Alcaligenes xylosoxidans cytochrome c' (Axcyt c') and of cardiolipin-bound horse heart cytochrome c (CL-hhcyt c) have been reported to bind NO to the "dark side" of the heme (i.e., as the proximal axial ligand) replacing the endogenous ligand His. Conversely, CL-free hhcyt c behaves as ferrous myoglobin by binding NO to the heme distal side, keeping His as the proximal axial ligand. Moreover, the ferrous derivative of CL-hhcyt c binds CO at the heme distal side, the proximal axial ligand being His. Furthermore, CL-hhcyt c shows peroxidase activity. In contrast, CL-free hhcyt c does not bind CO and does not show peroxidase activity. This suggests that heme-proteins may utilize both sides of the heme for ligand discrimination, which appears to be modulated allosterically. Here, structural and functional aspects of NO binding to ferrous Axcyt c' and (CL-)hhcyt c are reviewed.     

Fast folding of cytochrome c.

Native iso-2 cytochrome c contains two residues (His 18, Met 80) coordinated to the covalently attached heme. On unfolding of iso-2, the His 18 ligand remains coordinated to the heme iron, whereas Met 80 is displaced by a non-native heme ligand, His 33 or His 39. To test whether non-native His-heme ligation slows folding, we have constructed a double mutant protein in which the non-native ligands are replaced by asparagine and lysine, respectively (H33N,H39K iso-2). The double mutant protein, which cannot form non-native histidine-heme coordinate bonds, folds significantly faster than normal iso-2 cytochrome c: gamma = 14-26 ms for H33N,H39K iso-2 versus gamma = 200-1,100 ms for iso-2. These results with iso-2 cytochrome c strongly support the hypothesis that non-native His-heme ligation results in a kinetic barrier to fast folding of cytochrome c. Assuming that the maximum rate of a conformational search is about 10(11) s-1, the results imply that the direct folding pathway of iso-2 involves passage through on the order of 10(9) or fewer partially folded conformers.     

Kinetics of dithionite reduction of the heme nonapeptide of cytochrome c

The kinetics of dithionite reduction of the oxidized heme nonapeptide fragment of horse heart cytochrome c have been measured as a function of ionic strength at pH 7 and pH 9 by the stopped-flow technique. Dithionite concentration dependences indicate that the radical anion monomer, SO2-., is the active reductant. The pH 7 ionic strength dependence suggests that the heme peptide is reacting as a negatively charged molecule (its overall charge is calculated to be -1). Comparison of these results with the known rate of dithionite reduction of cytochrome c indicates that the heme nonapeptide has substantially greater inherent reactivity than cytochrome c, perhaps due to the greater accessibility of the heme.     

High-resolution three-dimensional structure of horse heart cytochrome c

The 1.94 A resolution three-dimensional structure of oxidized horse heart cytochrome c has been elucidated and refined to a final R-factor of 0.17. This has allowed for a detailed assessment of the structural features of this protein, including the presence of secondary structure, hydrogen-bonding patterns and heme geometry. A comprehensive analysis of the structural differences between horse heart cytochrome c and those other eukaryotic cytochromes c for which high-resolution structures are available (yeast iso-1, tuna, rice) has also been completed. Significant conformational differences between these proteins occur in three regions and primarily involve residues 22 to 27, 41 to 43 and 56 to 57. The first of these variable regions is part of a surface beta-loop, whilst the latter two are located together adjacent to the heme group. This study also demonstrates that, in horse cytochrome c, the side-chain of Phe82 is positioned in a co-planar fashion next to the heme in a conformation comparable to that found in other cytochromes c. The positioning of this residue does not therefore appear to be oxidation-state-dependent. In total, five water molecules occupy conserved positions in the structures of horse heart, yeast iso-1, tuna and rice cytochromes c. Three of these are on the surface of the protein, serving to stabilize local polypeptide chain conformations. The remaining two are internally located. One of these mediates a charged interaction between the invariant residue Arg38 and a nearby heme propionate. The other is more centrally buried near the heme iron atom and is hydrogen bonded to the conserved residues Asn52, Tyr67 and Thr78. It is shown that this latter water molecule shifts in a consistent manner upon change in oxidation state if cytochrome c structures from various sources are compared. The conservation of this structural feature and its close proximity to the heme iron atom strongly implicate this internal water molecule as having a functional role in the mechanism of action of cytochrome c.     

Strategic roles of axial histidines in structure formation and redox regulation of tetraheme cytochrome c3

Tetraheme cytochrome c 3 (cyt c 3) exhibits extremely low reduction potentials and unique properties. Since axial ligands should be the most important factors for this protein, every axial histidine of Desulfovibrio vulgaris Miyazaki F cyt c 3 was replaced with methionine, one by one. On mutation at the fifth ligand, the relevant heme could not be linked to the polypeptide, revealing the essential role of the fifth histidine in heme linking. The fifth histidine is the key residue in the structure formation and redox regulation of a c-type cytochrome. A crystal structure has been obtained for only H25M cyt c 3. The overall structure was not affected by the mutation except for the sixth methionine coordination at heme 3. NMR spectra revealed that each mutated methionine is coordinated to the sixth site of the relevant heme in the reduced state, while ligand conversion takes place at hemes 1 and 4 during oxidation at pH 7. The replacement of the sixth ligand with methionine caused an increase in the reduction potential of the mutated heme of 222-244 mV. The midpoint potential of a triheme H52M cyt c 3 is higher than that of the wild type by approximately 50 mV, suggesting a contribution of the tetraheme architecture to the lowering of the reduction potentials. The hydrogen bonding of Thr24 with an axial ligand induces a decrease in reduction potential of approximately 50 mV. In conclusion, the bis-histidine coordination is strategically essential for the structure formation and the extremely low reduction potential of cyt c 3.     

State of heme in heme c systems: Cytochrome c and heme c models

Polarized resonance Raman spectra of horse heart ferricytochrome c as a function of pH in the range 1.0–12, in the presence of the extrinsic ligands imidazole, cyanide, and azide, and in 4 M urea, are reported, as are resonance Raman spectra of heme undecapeptide in the presence of imidazole, pH 6.8 and pH 2.0, and with cyanide at pH 6.8. The range of investigation is 140–1700 cm^?1, using the 5145-, 4880-, and 4579-Å excitations. The spectra have been analyzed in terms of complexity, sensitivity, and the conformation-heme energetics of the systems. The state of heme in various forms is analyzed with regard to heme energetics, core size, nature of planarity, and coordination configuration. All low-spin forms of heme c systems, cytochrome c, and heme models are concluded to be hexacoordinated, in-plane heme iron systems. The effect of the location of the heme in the protein environment is found to be a slight expansion of the porphyrin core, ～0.01 Å, while the covalent linkage of heme to protein and a mixed nature of axial coordination configuration seem to have little effect on the energetics of the heme group. Complex formation with extrinsic ligand, imidazole, cyanide, or azide, results in a slight contraction of the heme core. The formation of cytochrome c form IV, the alkaline form, is shown to follow a process with apK _a of about 8.4, and similarly, acidic form II is created following the prior formation of an intermediate form with apK _a of about 3.6. The precursor to form IV is interpreted as containing perturbation of the pyrrol rings, whereas the precursor to the acidic form seems to reflect alteration of the energetics of the C_αC_{m α} structures of the heme group. The acidic form of heme undecapeptide is a hexacoordinated high-spin heme with an estimated displacement of 0.25 Å from the heme plane. The pH 2 form of cytochrome c is also a hexacoordinated high-spin form with two weak axial ligands, but iron is in the plane of the porphyrin ring.     

Q-Band resonance Raman investigation of turnip cytochrome f and Rhodobacter capsulatus cytochrome c1

The results of a comprehensive Q-band resonance Raman investigation of cytochrome c1 and cytochrome f subunits of bc1 and b6f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry.     

A spin label study of conformational changes in cytochrome c

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.     

A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.     

Replacement of putative axial ligands of heme iron in yeast cytochrome c1 by site-directed mutagenesis

The His-44 and Met-164 residues of yeast cytochrome c1 are evolutionally conserved and regarded as heme axial ligands bonding to the fifth and sixth coordination sites of the heme iron, which is directly involved in the electron transfer mechanism. Oligonucleotide-directed mutagenesis was used to generate mutant forms of cytochrome c1 of yeast having amino acid replacements of the putative axial ligands of the heme iron. When a cytochrome c1-deficiency yeast strain was transformed with a gene encoding the Phe-44, Tyr-44, Leu-164, or Lys-164 protein, none of these transformants could grow on the non-fermentable carbon source. These results suggest that the His-44 and Met-164 residues have a critical role in the function of cytochrome c1 in vivo, most probably as axial ligands of the heme iron. Further analysis revealed that the mutant yeast cells with the Phe-44, Tyr-44, or Leu-164 protein lacked the characteristic difference spectroscopic signal of cytochrome c1. However, in the Lys-164 mutant cells, partial recovery of the cytochrome c1 signal was observed. Moreover, the Lys-164 protein retained a low but significant level of succinate-cytochrome c reductase activity in vitro. The possibility that the nitrogen of Lys-164 served as the sixth heme ligand is discussed in comparison with cytochrome f of a photosynthetic electron-transfer complex, in which lysine has been proposed to be the sixth ligand.     

Crystal structures of an oxygen-binding cytochrome c from Rhodobacter sphaeroides

The photosynthetic bacterium Rhodobacter sphaeroides produces a heme protein (SHP), which is an unusual c-type cytochrome capable of transiently binding oxygen during autooxidation. Similar proteins have not only been observed in other photosynthetic bacteria but also in the obligate methylotroph Methylophilus methylotrophus and the metal reducing bacterium Shewanella putrefaciens. A three-dimensional structure of SHP was derived using the multiple isomorphous replacement phasing method. Besides a model for the oxidized state (to 1.82 A resolution), models for the reduced state (2.1 A resolution), the oxidized molecule liganded with cyanide (1. 90 A resolution), and the reduced molecule liganded with nitric oxide (2.20 A resolution) could be derived. The SHP structure represents a new variation of the class I cytochrome c fold. The oxidized state reveals a novel sixth heme ligand, Asn(88), which moves away from the iron upon reduction or when small molecules bind. The distal side of the heme has a striking resemblance to other heme proteins that bind gaseous compounds. In SHP the liberated amide group of Asn(88) stabilizes solvent-shielded ligands through a hydrogen bond.     

The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5

A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b(5) > His39Cys > His39Ser.     

Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase

The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.     

Multiple low spin forms of the cytochrome c ferrihemochrome. EPR spectra of various eukaryotic and prokaryotic cytochromes c.

1. Despite the same methionine-sulfur:heme-iron:imidazole-nitrogen hemochrome structure observed by x-ray crystallography in four of the seven c-type eukaryotic and prokaryotic cytochromes examined, and the occurrence of the characteristic 695 nm absorption band correlated with the presence of a methionine-sulfur:heme-iron axial ligand in all seven proteins, they fall into two distinct classes on the basis of their EPR and optical spectra. The horse, tuna, and bakers' yeast iso-1 cytochromes c have a predominant neutral pH EPR form with g1=3.06, g2=2.26, and g3=1.25, while the bakers' yeast iso-2 and Euglena cytochromes c, the Rhodospirillum rubrum cytochrome c2, and the Paracoccus denitrificans cytochrome c550 all have a predominant neutral pH EPR form with g1=3.2, g2=2.05, and g3=1.39. The ferricytochromes with g1=3.06 have a B-Q splitting that is approximately 150 cm-1 larger than the ferricytochromes with g1=3.2. 2. Each of the cytochromes displays up to four low spin EPR forms that are in pH-dependent equilibrium and can all be observed at near neutral pH. As the pH is raised the predominant neutral pH form is converted into two forms with g1=3.4 and g1=3.6, identified by comparsion with model compounds and other heme proteins as epsilon-amino:heme-iron:imidazole and bis-epsilon-amino:heme-iron ferrihemochromes, respectively. 3. The pK for the conversion of the predominant neutral pH EPR form into the alkaline pH forms is the same as the pK for the disappearance of the 695 nm absorption band for the cytochromes, even though these pK values range over 2 pH units. This confirms that the g1=3.06 and g1=3.2 forms contain the methionine-sulfur:heme-iron axial ligand while the g1=3.4 and the g1=3.6 forms do not. 4. At extremes of pH, the horse and bakers' yeast iso-1 proteins display several high and low spin forms that are identified, showing that a variety of protein-derived ligands will coordinate to the heme iron including methionine and cysteine sulfur, histidine imidazole, and lysine epsilon-amine. 5. The spectrum of horse cytochrome c with added azide, cyanide, hydroxide, or imidazole as axial ligands has also been examined. 6. From a comparison of the EPR and optical spectral characteristics of these groups of cytochromes with model compounds, it is suggested that the difference between them is due to a change in the hydrogen bonding or perhaps even in the protonation of N-1 of the heme iron-bound histidine imidazole.