Absorption Spectrum Of Cytochrome Research Articles

Integration of a Micro-Transparent heater-integrated electrochemical (μTHE) cell for investigation of the thermal stability of cytochrome c redox variants

Spectroelectrochemistry (SEC) is a powerful technique that combines spectroscopic and electrochemical properties to investigate the redox potential and spectral characterization of chemical or biological molecules. Recently, SEC has gained attention in quantitative analysis, particularly in exploring the role of redox-active proteins in electron transfer networks related to biological systems. The microfluidic technique is used to investigate the spectroelectrochemical properties of the redox potential of metalloproteins under accurate temperature environments. However, conventional heater systems suffer from long ramping times and the transparency of heat systems would influence the spectroscopic information. In this study, we fabricated a micro-transparent heater-integrated electrochemical (μTHE) cell. The μTHE cell allows for spectroelectrochemical studies to investigate the thermal stability of cytochrome c at specific temperatures and the transparent ITO heater integrated into the μTHE cell offers rapid and accurate temperature control during the measurements, avoiding interference with spectroscopic information. The μTHE cell could regulate the oxidation/reduction forms of cytochrome c by the electrochemical process, and the ITO heater provides the precise temperature control for the observation of the absorption spectrum of cytochrome c at different temperatures. By taking advantage of rapid heating and transparency, the μTHE cell offers a facile platform for investigating the spectroelectrochemical properties of redox-active molecules in a well-controlled environment.

Effect of red and near-infrared wavelengths on low-level laser (light) therapy-induced healing of partial-thickness dermal abrasion in mice

Low-level laser (light) therapy (LLLT) promotes wound healing, reduces pain and inflammation, and prevents tissue death. Studies have explored the effects of various radiant exposures on the effect of LLLT; however, studies of wavelength dependency in in vivo models are less common. In the present study, the healing effects of LLLT mediated by different wavelengths of light in the red and near-infrared (NIR) wavelength regions (635, 730, 810, and 980 nm) delivered at constant fluence (4 J/cm(2)) and fluence rate (10 mW/cm(2)) were evaluated in a mouse model of partial-thickness dermal abrasion. Wavelengths of 635 and 810 nm were found to be effective in promoting the healing of dermal abrasions. However, treatment using 730- and 980-nm wavelengths showed no sign of stimulated healing. Healing was maximally augmented in mice treated with an 810-nm wavelength, as evidenced by significant wound area reduction (p < 0.05), enhanced collagen accumulation, and complete re-epithelialization as compared to other wavelengths and non-illuminated controls. Significant acceleration of re-epithelialization and cellular proliferation revealed by immunofluorescence staining for cytokeratin-14 and proliferating cell nuclear antigen (p < 0.05) was evident in the 810-nm wavelength compared with other groups. Photobiomodulation mediated by red (635 nm) and NIR (810 nm) light suggests that the biological response of the wound tissue depends on the wavelength employed. The effectiveness of 810-nm wavelength agrees with previous publications and, together with the partial effectiveness of 635 nm and the ineffectiveness of 730 and 980 nm wavelengths, can be explained by the absorption spectrum of cytochrome c oxidase, the candidate mitochondrial chromophore in LLLT.

Inhibition of the yeast cytochrome bc1 complex by ilicicolin H, a novel inhibitor that acts at the Qn site of the bc1 complex.

Ilicicolin H is an antibiotic isolated from the "imperfect" fungus Cylindrocladium iliciola strain MFC-870. Ilicicolin inhibits mitochondrial respiration by inhibiting the cytochrome bc(1) complex. In order to identify the site of ilicicolin action within the bc(1) complex we have characterized the effects of ilicicolin on the cytochrome bc(1) complex of Saccharomyces cerevisiae. Ilicicolin inhibits ubiquinol-cytochrome c reductase activity of the yeast bc(1) complex with an IC(50) of 3-5 nM, while 200-250 nM ilicicolin was required to obtain comparable inhibition of the bovine bc(1) complex. Ilicicolin blocks oxidation-reduction of cytochrome b through center N of the bc(1) complex and promotes oxidant-induced reduction of cytochrome b but has no effect on oxidation of ubiquinol through center P. These results indicate that ilicicolin binds to the Qn site of the bc(1) complex. Ilicicolin induces a blue shift in the absorption spectrum of ferro-cytochrome b, and titration of the spectral shift indicates binding of one inhibitor molecule per Qn site. The effects of ilicicolin on electron transfer reactions in the bc(1) complex are similar to those of antimycin, another inhibitor that binds to the Qn site of the bc(1) complex. However, because the two inhibitors have different effects on the absorption spectrum of cytochrome b, they differ in their mode of binding to the Qn site.

Far-Infrared Absorption Measurements of Polypeptides and Cytochrome c by THz Radiation

Abstract Pulsed terahertz (THz) radiation and black-body radiation are applied to measure far infrared (FIR) absorption spectra of polypeptides and cytochrome c in the wavenumber region from 7 cm-1 to 160 cm-1. In the region from 7 cm-1 to 55 cm-1, FIR absorption cross sections of polyglycine and poly-L-alanine in powder are greater than those of glycine and L-alanine in powder. On the other hand, FIR absorption spectra of cytochrome c in lyophilized powder show little dependence on protein structures. The structures of biopolymers are investigated by mid-IR absorption (polypeptides and cytochrome c) and by resonance Raman scattering (cytochrome c). FIR spectral features of biopolymers in the THz frequency region are qualitatively discussed in terms of density of states and homogeneous/inhomogeneous broadening.

Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression.

A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.

Evidence for an essential histidine residue in the ascorbate-binding site of cytochrome b561.

Cytochrome b(561) mediates equilibration of the ascorbate/semidehydroascorbate redox couple across the membranes of secretory vesicles. The cytochrome is reduced by ascorbic acid and oxidized by semidehydroascorbate on either side of the membrane. Treatment with diethyl pyrocarbonate (DEPC) inhibits reduction of the cytochrome by ascorbate, but this activity can be restored by subsequent treatment with hydroxylamine, suggesting the involvement of an essential histidine residue. Moreover, DEPC inactivates cytochrome b(561) more rapidly at alkaline pH, consistent with modification of a histidine residue. DEPC does not affect the absorption spectrum of cytochrome b(561) nor does it change the midpoint reduction potential, confirming that histidine modification does not affect the heme. Ascorbate protects the cytochrome from inactivation by DEPC, indicating that the essential histidine is in the ascorbate-binding site. Further evidence for this is that DEPC treatment inhibits oxidation of the cytochrome by semidehydroascorbate but not by ferricyanide. This supports a reaction mechanism in which ascorbate loses a hydrogen atom by donating a proton to histidine and transferring an electron to the heme.

Non-Contact Measurement of Adsorbed Cytochrome c with Optical Waveguide Spectrometry; The Effect of Distance between Waveguide and Protein on the Spectral Sensitivity

Abstract Optical waveguide spectrometry with non-contact conditions was performed with the aim of widening the choice of substrate material. The visible absorption spectrum of cytochrome c (cyt.c) adsorbed on a quartz glass or gold surface was detected with the evanescent wave from a co-faced waveguide. A clear correlation was observed between absorbance of cyt.c and the gap between the waveguide surface and the protein.

The Second Derivative Electronic Absorption Spectrum of Cytochrome c Oxidase in the Soret Region

The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400–500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600 cm −1, was observed in the direct absorption spectrum of the a 2+ a 3 3+·HCOO − form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a 3. Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A 3 chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.

Characterization of Quinohemoprotein Amine Dehydrogenase from Pseudomonas putida.

Quinohemoprotein amine dehydrogenase (AMDH) was purified and crystallized from the soluble fraction of Pseudomonas putida IFO 15366 grown on n-butylamine medium. AMDH gave a single component in analytical ultracentrifugation showing an intrinsic sedimentation coefficient of 5.8s. AMDH showed a typical absorption spectrum of cytochrome c showing maxima at 554, 522, 420, and 320 nm in the reduced form and one peak at 410 nm, a shoulder at 350 nm, and a broad hill around 530 nm in the oxidized form. The oxidized enzyme was specifically reduced by the addition of amine substrate. AMDH was composed of three different subunits, 60, 40, and 20 kDa, with the total molecular weight of 120,000. Two moles of heme c were detected per mole of AMDH and the 60-kDa subunit was found to be the heme c-carrying subunit. By redox-cycling quinone staining, a positive reaction band corresponding to the 20-kDa subunit was detected after developed by SDS-PAGE, but the 20 kDa band was scarcely stained by conventional protein staining. Only a silver staining method was possible to detect the subunit after the protein was developed by SDS-PAGE. p-Nitrophenylhydrazine-inhibited AMDH was dissociated into subunits and the 20-kDa subunit showed an absorption maximum at 455 nm, indicating Schiff base formation between the carbonyl cofactor in AMDH and the carbonyl reagent. Thus, AMDH is different from nonheme quinoprotein methylamine dehydrogenase and aromatic amine dehydrogenase in many respects. The presence of an azurin-like blue protein was identified and purified from the same cell-free extract of P. putida as AMDH was purified. The blue protein was reduced specifically during AMDH reaction, suggesting that the blue protein is the direct electron acceptor in amine oxidation. The amine oxidation system was reconstituted successfully only by AMDH, the blue protein, and the cytoplasmic membranes of the organism. The function of the 40-kDa subunit is unknown at the moment. The properties of AMDH were compared with other bacterial amine dehydrogenases so far reported.

Accessibility of Tyrosine Residues in Cytochrome P-450scc (CYP11A1)

Cytochrome P-450scc (CYP11A1) is known to exist in various conformational states. To study the accessibility of tyrosine residues to the solvent, second and fourth derivatives of cytochrome absorption spectra in the ultraviolet region were used. The measurements were carried out in the temperature range -10 to 40 °C and at two pH values (6.8 and 7.4). Our results indicate that the tyrosine residues of this enzyme are less accessible at higher temperatures as well as at higher pH, that is at conditions where the conformational equilibrium shifts to low-spin Fe(III) form(s). In other words, a more compact structure is attributable to the low-spin Fe(III) form(s) of P-450scc.

Function and morphology of the retinal pigment epithelium after light-induced damage.

The purpose of this study was to determine the threshold energy for light-induced functional damage of the retinal pigment epithelium at various wavelengths. Retinas of 58 pigmented and 21 albino rabbits were exposed to low intensity broadband blue light (400-520 nm), yellow light (510-740 nm), and narrowband blue light (408, 417, 439, 455, 485, 501 nm, respectively; deltalambda = 10-13 nm). The intensity values were 50, 280, and 5 mW x cm (-2), respectively, and the illumination time was 0.5 up to 5 h. The cumulative dose of light energy was calculated from these data (J x cm(-2)). The blood-retinal barrier dysfunction was evaluated in vivo using fluorophotometry to measure the leakage of fluorescein into the vitreous after intravenous injection and in vitro using light and electron microscopy after an in vivo intraarterial injection of horseradish peroxidase (HRP). The threshold energy for fluorescein leakage was 50 J x cm (-2) for blue light and 1,600 J x cm(-2) for yellow light. After broadband blue light exposure, the HRP reaction product was seen in the cytoplasm of the retinal pigment epithelium (RPE) cells and in the subretinal space but only if fluorescein leakage had been observed. Threshold energy and fluorescein leakage as a function of light energy were similar for albino and pigmented rabbits (P > 0.5). Only after yellow light exposure in excess of 3,700 J x cm(-2) was fluorescein leakage found. In that case complete disruption of the RPE was seen, but no HRP was observed in the RPE cytoplasm. Of the narrow-band blue light exposures, only that at lambda = 418 nm caused a significant increase in fluorescein leakage; the threshold energy was 18 J x cm(- 2). Blue light was found to be at least 30 times more efficient than yellow light in causing dysfunction of the blood-retinal barrier. The most efficient wavelength was 418 nm, corresponding with the absorption spectrum of cytochrome c oxidase. Melanin seemed to play no role. The presence or absence of melanin in the RPE appeared to have no influence on the threshold energy.

Oxygen-dependent reversible inhibition of mitochondrial respiration by nitric oxide.

Effects of nitric oxide (NO) and NO generating agents, on the electron transport system of mitochondria were examined in a study of the mechanism and physiological importance of NO in energy metabolism. In the presence of various substrates, uncoupled respiration was inhibited by NO in manner which was both dose- and oxygen tension-dependent. Simultaneously measuring changes in cytochrome absorption spectra and respiration showed that the site of action of NO is cytochrome oxidase. Similar inhibition was also brought about by 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC 18), an NO donor. Electron paramagnetic resonance (EPR) analysis revealed that inhibition of uncoupled respiration occurred only during the presence of NO in the reaction mixture. The inhibitory effect of NO was increased significantly by lowering the concentration of mitochondrial protein. No appreciable inhibition of respiration was observed in the presence of 3-morpholinosydnonimine (SIN-1), a peroxynitrite anion (ONOO-) generating reagent, but inhibition did occur in the presence of superoxide dismutase (SOD). These results indicate that NO reversibly interacts with mitochondria at complex IV thereby inhibiting respiration particularly under physiologically low oxygen tension and that de novo generated ONOO may have no significant effect under the present experimental conditions.

Purification of a membrane-bound sorbitol dehydrogenase fromGluconobacter suboxydans

A sorbitol dehydrogenase was purified from the membrane fraction of Gluconobacter suboxydans KCTC 2111 (= ATCC 621) by chromatography on CM-, DEAE-, Mono S and Superose 12 columns. The purified enzyme showed a single activity band upon nondenaturing polyacrylamide gel electrophoresis (PAGE) and three subunits of 75, 50 and 14 kDa upon SDS-PAGE. When purified preparations of the enzyme were reconstituted with pyrroloquinoline quinone (PQQ), the specific enzyme activity was significantly increased (up to 9-fold). The absorption spectrum of purified sorbitol dehydrogenase in the reduced state exhibited three absorption maxima (417, 522 and 552 nm) which is in accordance with the typical absorption spectrum of cytochrome c. The 50 kDa subunit appeared as a red band on unstained SDS-gels suggesting its identity as a cytochrome. Fluorescence spectra of extracts from purified sorbitol dehydrogenase showed an excitation maximum at 370 nm and an emission maximum at 465 nm, which conformed to those of authentic PQQ. The purified enzyme showed a rather broad substrate specificity with significant activity toward D-mannitol (68%) and D-ribitol (70%) as well as D-sorbitol (100%). The PQQ-dependent sorbitol dehydrogenase described in this study is clearly different from the FAD-dependent sorbitol dehydrogenase from G. suboxydans var. α IFO 3254 strain in its cofactor requirement and substrate specificity.

Purification of a membrane-bound sorbitol dehydrogenase from Gluconobacter suboxydans

A sorbitol dehydrogenase was purified from the membrane fraction of Gluconobacter suboxydans KCTC 2111 (= ATCC 621) by chromatography on CM-, DEAE-, Mono S and Superose 12 columns. The purified enzyme showed a single activity band upon nondenaturing polyacrylamide gel electrophoresis (PAGE) and three subunits of 75, 50 and 14 kDa upon SDS-PAGE. When purified preparations of the enzyme were reconstituted with pyrroloquinoline quinone (PQQ), the specific enzyme activity was significantly increased (up to 9-fold). The absorption spectrum of purified sorbitol dehydrogenase in the reduced state exhibited three absorption maxima (417, 522 and 552 nm) which is in accordance with the typical absorption spectrum of cytochrome c. The 50 kDa subunit appeared as a red band on unstained SDS-gels suggesting its identity as a cytochrome. Fluorescence spectra of extracts from purified sorbitol dehydrogenase showed an excitation maximum at 370 nm and an emission maximum at 465 nm, which conformed to those of authentic PQQ. The purified enzyme showed a rather broad substrate specificity with significant activity toward D-mannitol (68%) and D-ribitol (70%) as well as D-sorbitol (100%). The PQQ-dependent sorbitol dehydrogenase described in this study is clearly different from the FAD-dependent sorbitol dehydrogenase from G. suboxydans var. α IFO 3254 strain in its cofactor requirement and substrate specificity.

A protein which binds preferentially to single-stranded core sequence of autonomously replicating sequence is essential for respiratory function in mitochondrial of Saccharomyces cerevisiae.

From yeast nuclear extract, we have identified several DNA-protein complexes using the T-rich strand of core consensus sequence of autonomously replicating sequence by gel shift assay. One of them showed preferential binding to the T-rich sequence of the DNA. We have partially purified a protein constituent of this complex and cloned its gene. The gene has an open reading frame encoding a protein of 380 amino acids (M(r) = 42,100) which is processed to a mature protein of 371 amino acids (M(r) = 40,900). The protein has neither significant amino acid homology with any previously reported proteins nor characteristic motifs. A putative HAP2/HAP3/HAP4 binding sequence was found at about 1 kilobase upstream of the gene. Disruption of the chromosomal gene revealed that the gene was neither essential for cell viability nor involved in DNA replication, but was essential for mitochondrial respiratory function. We therefore named the gene MRF1 for mitochondrial respiratory function 1. In a mrf1 null mutant the absorption spectra of cytochromes b, a, and a3 were undetectable, although mitochondrial DNA and protein synthesis in mitochondria were intact. Antibodies against MRF1 detected the antigen localized predominantly in the nucleus in vivo. These results suggest that MRF1 is a transcriptional regulatory protein of some genes whose products are necessary for the functional assembly of mitochondrial respiratory proteins.

Evidence for a band III analogue in the near-infrared absorption spectra of cytochrome [formula omitted] oxidase

Ground state near-infrared absorption spectra of fully reduced unliganded and fully reduced CO ( a 2+ Cu A + a 3 2+-CO Cu B + ) cytochrome c oxidase were investigated. Flash-photolysis time-resolved absorption difference spectra of the mixed-valence ( a 3+ Cu A 2+ a 3 2+-CO Cu B + ) and the fully reduced CO complexes were also studied. A band near 785 nm (ε ∼50 M −1cm −1) was observed in the fully reduced unliganded enzyme and the CO photoproducts. The time-resolved 785 nm band disappeared on the same timescale ( t 1 2 ∼7 ms ) as CO recombined with cytochrome a 3 2+ . This band, which is attributed to the unliganded five coordinate ferrous cytochrome a 3 2+ , has some characteristics of band III in deoxy-hemoglobin and deoxy-myoglobin. A second band was observed at ∼710 nm (ε ∼80 M −1cm −1) in the fully reduced unliganded and the fully reduced CO complexes. This band, which we assign to the low spin ferrous cytochrome a , appears to be affected by the ligation state at the cytochrome a 3 2+ site.

Analysis of cytochromes and co-enzymes Q in the yeast genus Candida.

The cytochrome absorption spectra of whole cell pastes and the co-enzyme Q system were analyzed in eighteen species in the yeast genera Candida and Pichia. C. silvanorum, C. entomophila and C. homilentoma had Co-Q 8 as the major component and Co-Q 6, 7 and 9 as minor components. The other yeasts had Co-Q 9 as the major component with Co-Q 8 as the first minor component associated with an additional Co-Q 7 in five species (C. shehatae, C. naeodendra, C. butyri, C. tenuis and C. fluviotilis) or Co-Q 10 in three others (C. diddensii, C. polymorpha and C. atmosphaerica). Pichia stipitis may no longer be considered as the perfect form of C. shehatae.These results confirmed that Co-Q system composition is a valuable tool for the chemotaxonomy of yeasts when used in conjunction with spectrophotometric analysis of cytochromes and other techniques such as GC content, DNA/DNA hybridization or serology.

Purification of Vibrio fischeri nitrite reductase and its characterization as a hexaheme c-type cytochrome

Dissimilatory nitrite reductase was isolated from anaerobically nitrate-grown Vibrio fischeri cells and purified to electrophoretic homogeneity. The enzyme catalyzes the six-electron reduction of nitrite to ammonia. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under either nonreducing or reducing conditions, the purified nitrite reductase migrated as a single protein band of M r 57,000. Gel filtration chromatography revealed a native molecular weight of 58,000, indicating the enzyme as isolated to be present in the monomeric form. Purified nitrite reductase exhibited typical c-type cytochrome absorption spectra with the reduced α-band at 552.5 nm. Heme content analysis using the purified preparation indicated the enzyme to contain 5.5 heme c groups per molecule. Iron analysis showed the presence of 5.62 g iron atoms per mole of enzyme and no nonheme irons were detected. These results clearly indicate that, similar to the dissimilatory nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes, and Escherichia coli, the V. fischeri nitrite reductase is a hexaheme c-type cytochrome. Amino acid composition of V. fischeri also revealed close similarities to those of the other three hexaheme nitrite reductases previously studied. Based on this information, it is concluded that the four ammonia-forming, dissimilatory nitrite reductases isolated to date represent a homologous group of proteins with the distinct property of being hexaheme c-type cytochromes.

Spectrophotometric analysis of yeasts. Cytochrome spectra of some Candida and related taxa.

Whole-cell pastes of eight Candida species and their suggested teleomorph counterparts in the genera Pichia and Issatchenkia analyzed were spectrophotometrically. All of these yeasts, included in the two very closely related groups krusei and norvegensis, showed strong similarities in their low-temperature cytochrome absorption spectra, especially in two typical peaks, generally at 597-597.5nm and 605-606.5nm or in some cases at 593.5-594.5nm and 601.5-602nm, in the spectral region of the α maximum absorption of cytochrome aa3. In addition, the two species of each assumed pair of perfect and imperfect yeasts, I. orientalis/C. krusei, I.occidentalis/C. sorbosa, P. fermentans/C. lambica, P. nakasei/C. citrea or P. norvegensis/C. norvegensis were indistinguishable. Cytochrome spectra also indicated that I. terricola may no longer be considered a teleomorph of C. diversa.

Isolation and crystallization of high molecular weight cytochrome from Desulfovibrio valgaris Hildenborough

High molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough has been isolated, purified and crystallized. The molecular weight was estimated to be 75 000 as the mean value from the results of gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains 16 c-type heme groups per molecule. The purified high molecular weight cytochrome c shows typical c-type cytochrome absorption spectra, with an α-peak at 553.2 nm ( ϵ 553.2 mM = 428) in the ferro form. The amino acid composition shows that the protein contains a sufficient number of cysteine and histidine residues to account for the high content of heme groups. The amino acid composition of D. vulgaris Hildenborough high molecular weight cytochrome c has no similarities to the D. desulfuricans hexahemoprotein nitrite reductase. The high content of the heme groups might suggest that the cytochrome has an alternate intrinsic biological function. Crystals of the high molecular weight cytochrome c have been grown from solutions of poly(ethylene glycol) 1000 or 2-methyl-2,4-pentanediol. The crystals are in space group P6 2 or P6 4 with unit cell dimensions a = b = 227.8, c = 105.7 Å and γ = 120°. A large unit cell volume of 4.75 · 10 6 Å 3 suggests that there are four or five protein molecules per asymmetric unit. They diffract to better than 4.0 Å resolution and appear to be resistant to radiation damage.