Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli

c-Type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 (c-b562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.     

Stimulation of tetrapyrrole formation in Rhizobium japonicum by restricted aeration.

Cultures of Rhizobium japonicum were grown with vigorous aeration to stationary phase and were then incubated under restricted aeration for several days. Under these "microaerobic" conditions, cellular heme content increased 10-fold, and visible amounts of porphyrins were released into the culture medium. The two predominant porphyrins produced were identified, on the basis of their spectrophotometric and chromatographic properties, as protoporphyrin and coproporphyrin. The cytochrome complement of microaerobic cells partially resembled that of the symbiotic bacteria in that cytochromes alpha-alpha3 were absent and a CO-binding cytochrome 552 was present. During the period of restricted aeration, at the time that the heme content was increasing, there was a similar 10-fold increase in the activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase. However, during the same period, the activity of succinyl thiokinase (an enzyme that is required in large amounts whether or not heme is being produced) increased only twofold. These results suggest that reduced oxygen tension may play a role in inducing heme synthesis necessary for leghemoglobin formation and bacterial differentiation in soybean root nodules.     

Crystal structure of oxidized Bacillus pasteurii cytochrome c553 at 0.97-A resolution

This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolated from a Gram-positive bacterium. Bacillus pasteurii cytochrome c(553), characterized by a low reduction potential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes, is a useful case study for understanding the structure-function relationships for this class of electron-transfer proteins. Diffraction data on a single crystal of cytochrome c(553) were obtained using synchrotron radiation at 100 K. The structure was determined at 0.97-A resolution using ab initio phasing and independently at 1.70 A in an MAD experiment. In both experiments, the structure solution exploited the presence of a single Fe atom as anomalous scatterer in the protein. For the 0.97-A data, the phasing was based on a single data set. This is the most precise structure of a heme protein to date. The crystallized cytochrome c(553) contains only 71 of the 92 residues expected from the intact protein sequence, lacking the first 21 amino acids at the N-terminus. This feature is consistent with previous evidence that this tail, responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purification procedure. The heme prosthetic group in B. pasteurii cytochrome c(553) is surrounded by three alpha-helices in a compact arrangement. The largely exposed c-type heme group features a His-Met axial coordination of the Fe(III) ion. The protein is characterized by a very asymmetric charge distribution, with the exposed heme edge located on a surface patch devoid of net charges. A structural search of a representative set of protein structures reveals that B. pasteurii cytochrome c(553) is most similar to Pseudomonas cytochromes c(551), followed by cytochromes c(6), Desulfovibrio cytochrome c(553), cytochromes c(552) from thermophiles, and cytochromes c from eukaryotes. Notwithstanding a low sequence homology, a structure-based alignment of these cytochromes shows conservation of three helical regions, with different additional secondary structure motifs characterizing each protein. In B. pasteurii cytochrome c(553), these motifs are represented by the shortest interhelix connecting fragments observed for this group of proteins. The possible relationships between heme solvent accessibility and the electrochemical reduction potential are discussed.     

Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes

Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.     

Cytochrome and Ubiquinone Patterns During Growth of Azotobacter vinelandii

After synthesis during the early log phase, the concentrations of ubiquinone and cytochromes did not vary during the growth cycle of Azotobacter vinelandii, when grown with either high or low aeration on nitrogen-free or urea-containing media. The level of aeration had no effect on the concentrations of the electron carriers synthesized, but affected the growth rate. On urea-containing medium, the concentration of cytochrome a(2) was low, but it was synthesized at a linear rate when the bacteria were transferred to nitrogen-free medium. A. vinelandii was shown to utilize sufficient medium urea to account for all of the cell nitrogen. Growth on urea-containing medium with an oxygen atmosphere resulted in low growth yields, and cytochromes c(4) + c(5) were not synthesized; the concentrations of ubiquinone and cytochromes b(1), a(1), and a(2) were only 12 to 18% of the values for growth on nitrogen-free medium with high aeration.     

Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c1.

Cytochrome c reductase purified from the trypanosomatid Crithidia fasciculata retained antimycin A sensitivity and catalyzed the reduction of horse heart ferricytochrome c in the presence of reduced coenzyme Q10. The complex contained heme b and heme c1 in a ratio of 2:1. Nine major protein bands ranging in size from 55.3 to approximately 12.8 kDa were resolved by SDS-polyacrylamide gel electrophoresis. A 31.6-kDa protein was identified as cytochrome c1 by the presence of a covalently attached heme. A red shift in the alpha-absorbance band of the cytochrome c1 absolute absorbance spectrum, difference absorbance spectrum, and pyridine ferrohemochrome absorbance spectrum suggested that the heme prosthetic group of C. fasciculata cytochrome c1 is bound to the apoprotein through only one thioether bond. A fragment of the cytochrome c1 gene was amplified from C. fasciculata, Trypanosoma brucei, Leishmania tarentolae, and Bodo caudatus. The deduced heme binding site sequence of each of these kinetoplastid species, Phe-Ala-Pro-Cys-His, contains a phenylalanine rather that a cysteine at the first position so that only one thioether bond can be formed between heme and apoprotein. This phenylalanine substitution and the presence of a conserved proline in the sequence may represent compensatory changes that are necessary for optimal interaction of the cytochromes c1 with the atypical cytochromes c of these species.     

Cytochrome c1 of bakers' yeast. II. Synthesis on cytoplasmic robosomes and influence of oxygen and heme on accumulation of the apoprotein.

In the preceding paper (Ross, E., and Schatz, G. (1976) J. Biol. Chem. 251, 1991-1996) yeast cytochrome c1 was characterized as a 31,000 dalton polypeptide with a covalently bound heme group. In order to determine the site of translation of this heme-carrying polypeptide, yeast cells were labeled with [H]leu(be under the following conditions: (a) in the absence of inhibitors, (b) in the presence of acriflavin (an inhibitor of mitochondrial translation), or (c) in the presence of cycloheximide (an inhibitor of cytoplasmic translation). The incorporation of radioactivity into the hemeprotein was measured by immunoprecipitating it from mitochondrial extracts and analyzing it by dodecyl sulfate-polyacrylamide gel electrophoresis. Label was incorporated into the cytochrome c1 apoprotein only in the presence of acriflavin or in the absence of inhibitor, but not in the presence of cycloheximide. Cytochrome c1 is thus a cytoplasmic translation product. This conclusion was further supported by the demonstration that a cytolasmic petite mutant lacking mitochondrial protein synthesis still contained holocytochrome c1 that was indistinguishable from cytochrome c1 of wild type yeast with respect to molecular weight, absorption spectru, the presence of a covalently bound heme group, and antigenic properties. Cytochrome c1 in the mitochondria of the cytoplasmic petite mutant is firmly bound to the membrane, and its concentration approaches that typical of wild type mitochondria. However, its lability to proteolysis appeared to be increased. A mitochondrial translation product may thus be necessary for the correct conformation or orientation of cytochrome c1 in the mitochondrial inner membrane. Accumulation of cytochrome c1 protein in mitochondria is dependent on the abailability of heme. This was shown with a delta-aminolevulinic acid synthetase-deficient yeast mutant which lacks heme and any light-absorbing peaks attributable to cytochromes. Mitochondria from mutant cells grown without added delta-aminolevulinic acid contained at least 20 times less protein immunoprecipitable by cytochrome c1-antisera than mitochondria from cells grown in the presence of the heme precursor. Similarly, the respiration-deficient promitochondria of anaerobically grown wild type cells are almost completely devoid of material cross-reacting with cytochrome c1-antisera. A 105,000 X g supernatant of aerobically grown wild type cells contains a 29,000 dalton polypeptide that is precipitated by cytochrome c1-antiserum but not by nonimmune serum. This polypeptide is also present in high speed supernatants from the heme-deficient mutant or from anaerobically gorwn wild type cells. The possible identity of this polypeptide with soluble apocytochrome c1 is being investigated.     

Effect of aerobic growth conditions on the soluble cytochrome content of the purple phototrophic bacterium Rhodobacter sphaeroides: induction of cytochrome c554

When grown anaerobically in the light, Rhodobacter sphaeroides contains appreciable quantities of cytochromes c2 and c', but smaller amounts of other soluble cytochromes such as cytochrome c551.5, cytochrome c554, and an oxygen-binding heme protein. When R. sphaeroides is mass cultured aerobically in the dark to stationary phase, the content of cytochrome c2 does not change appreciably, whereas cytochrome c554 is approximately 8-fold more abundant, cytochrome c' is at least 10-fold less abundant, and cytochrome c551.5 is fivefold lower than in the phototrophically grown cells. These observations confirm previous literature reports that in this organism a cytochrome c553 (or c554 in our experience) is more abundant when cells are grown aerobically. Furthermore, the aerobic cytochrome c554 is positively identified with the previously characterized minor cytochrome c554 component of anaerobic photosynthetic cells. Preliminary sequence results show that cytochrome c554 is a member of the cytochrome c' structural family, but differs from normal cytochromes c' in having a methionine sixth ligand to the heme. The levels of electron carrier proteins of low redox potential had previously been reported to be less in aerobic than in photoheterotrophic cells and we have verified that observation for the specific examples of cytochromes c' and c551.5. The oxygen binding heme protein, SHP, is not induced by aerobic growth.     

Biochemical requirements for the maturation of mitochondrial c-type cytochromes

Cytochromes c are metalloproteins that function in electron transfer reactions and contain a heme moiety covalently attached via thioether linkages between the co-factor and a CXXCH motif in the protein. Covalent attachment of the heme group occurs on the positive side of all energy-transducing membranes (bacterial periplasm, mitochondrial intermembrane space and thylakoid lumen) and requires minimally: 1) synthesis and translocation of the apocytochromes c and heme across at least one biological membrane, 2) reduction of apocytochromes c and heme and maintenance under a reduced form prior to 3) catalysis of the heme attachment reaction. Surprisingly, the conversion of apoforms of cytochromes c to their respective holoforms occurs through at least three different pathways (systems I, II and III). In this review, we detail the assembly process of soluble cytochrome c and membrane-bound cytochrome c1, the only two mitochondrial c-type cytochromes that function in respiration. Mitochondrial c-type cytochromes are matured in the intermembrane space via the system I or system III pathway, an intriguing finding considering that the biochemical requirements for cytochrome c maturation are believed to be common regardless of the energy-transducing membrane under study.     

Cytochrome c biogenesis in mitochondria--Systems III and V

In c-type cytochromes, heme becomes covalently attached to the polypeptide chain by a reaction between the vinyl groups of the heme and cysteine thiols from the protein. There are two such cytochromes in mitochondria: cytochrome c and cytochrome c(1). The heme attachment is a post-translational modification that is catalysed by different biogenesis proteins in different organisms. Three types of biogenesis system are found or predicted in mitochondria: System I (the cytochrome c maturation system); System III (termed holocytochrome c synthase (HCCS) or heme lyase); and System V. This review focuses primarily on cytochrome c maturation in mitochondria containing HCCS (System III). It describes what is known about the enzymology and substrate specificity of HCCS; the role of HCCS in human disease; import of HCCS into mitochondria; import of apocytochromes c and c(1) into mitochondria and the close relationships with HCCS-dependent heme attachment; and the role of the fungal cytochrome c biogenesis accessory protein Cyc2. System V is also discussed; this is the postulated mitochondrial cytochrome c biogenesis system of trypanosomes and related organisms. No cytochrome c biogenesis proteins have been identified in the genomes of these organisms whose c-type cytochromes also have a unique mode of heme attachment.     

MauG,a novel diheme protein required for tryptophan tryptophylquinone biogenesis

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits. None of these gene products have been previously isolated. One of these, mauG, exhibits sequence similarity to diheme cytochrome c peroxidases and is required for the synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group of MADH. A system was developed for the homologous expression of MauG in P. denitrificans. Its signal sequence was correctly processed, and it was purified from the periplasmic cell fraction. The protein contains two covalent c-type hemes, as predicted from the deduced sequence. EPR spectroscopy reveals that the protein as isolated possesses about equal amounts of one high-spin heme with axial symmetry and one low-spin heme with rhombic symmetry. The low-spin heme contains a major and minor component suggesting a small degree of heme heterogeneity. The high-spin heme and the major low-spin heme component each exhibit resonances that are atypical of c-type hemes and dissimilar to those reported for diheme cytochrome c peroxidases. MauG exhibited only very weak peroxidase activity when assayed with either c-type cytochromes or o-dianisidine as an electron donor. Fully reduced MauG was shown to bind carbon monoxide and could be reoxidized by oxygen. The relevance of these unusual properties of MauG is discussed in the context of its role in TTQ biogenesis.     

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.     

Biosynthesis of glycoproteins in Candida albicans: Processing of a Man6-GlcNAc2-Asn oligosaccharide by membrane fractions

Abstract Bacillus subtilis can synthesise cytochromes containing a -, b -, c - and d -type heme. The biosynthetic pathways of these heme prosthetic groups were investigated by using strains blocked in uroporphyrinogen III synthesis from porphobilinogen or in heme b (protoheme IX) synthesis from uroporphyrinogen III. The results strongly suggest that heme a and heme d are both synthesised from heme b (protoheme IX). They also indicate that B. subtilis contains a novel ferrochelatase involved in the synthesis of siroheme.     

Recent studies of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of Escherichia coli when the bacteria are grown under conditions of high aeration. The oxidase is a ubiquinol oxidase and reduces molecular oxygen to water. Electron transport through the enzyme is coupled to the generation of a protonmotive force. The purified cytochrome o complex contains four or five subunits, two protoheme IX (heme b) prosthetic groups, plus at least one Cu. The subunits are all encoded by the cyo operon. Sequence comparisons show that the cytochrome o complex is closely related to the aa3-type cytochrome c oxidase family. Gene fusions have been used to define the topology of each of the gene products. Subunits I, II, III and IV are proposed to have 15, 2, 5 and 3 transmembrane spans, respectively. The fifth gene product (cyoE) encodes a protein with 7 membrane spanning segments, and this may also be a subunit of this enzyme. Fourier transform infrared spectroscopy has been used to monitor CO bound in the active site where oxygen is reduced. These data provide definitive proof that the cytochrome o complex has a heme-copper binuclear center, similar to that present in the aa3-type cytochrome c oxidases. Site-directed mutagenesis is being utilized to define which amino acids are ligands to the heme iron and copper prosthetic groups.     

Nitrate Reductase and Soluble Cytochrome c in Spirillum itersonii

The addition of nitrate to cultures of Spirillum itersonii incubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 x g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome c was increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome c rose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome c in response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome c does not require the presence of a functional nitrate reductase.     

Regulation of calbindin-D9k expression by 1,25-dihydroxyvitamin D(3) and parathyroid hormone in mouse primary renal tubular cells

Membranes of the obligate methylotroph Methylobacillus flagellatus KT contained hemes B, O, and C and cytochromes b, o, and c both in batch and in continuous cultures. Neither heme A nor heme D was detected in the membranes. The cytochromes o and bb were the main components reversibly binding carbon monoxide (CO) in the terminal part of the respiratory chain. The alpha-region and especially the alpha-peaks at 568 and 573 nm and the alpha-troughs at 586 and 592 on the CO-difference spectra were diagnostic for the cytochromes o and bb, respectively. The cytochrome o content increased up to 1.8 times upon increasing the dilution rate of the culture from 0.15 to 0.55 h(-1) under methanol limitation. By contrast, the level of the CO-binding cytochrome bb was not affected by methanol concentration but its content increased up to 1.9 times when the level of oxygen decreased from 95 to 21 microM under the constant dilution rate (mu = 0.55 h(-1)). The maximum ratio between the cytochromes o and bb reached 2 during continuous cultivation under methanol-limited conditions (mu = 0.55 h(-1)), whereas the minimum ratio between them was about 0.7 during batch cultivation at stationary phase of growth. The synthesis of the CO-binding cytochrome bb but not of the cytochrome o in M. flagellatus KT was assumed to depend on the ambient redox potential of the medium. The cytochrome o synthesis was supposed to depend on the transmembrane gradient of protons (Delta(mu)H+).     

Up-regulation of heme biosynthesis during differentiation of Neuro2a cells

Heme is an iron-containing tetrapyrrole molecule that functions as a prosthetic group for proteins such as mitochondrial respiratory enzymes. Several studies have suggested that heme has essential functions in the construction and maintenance of the nervous system. In this study, the contents of three biologically important forms of heme (types a, b, and c) and the expression of heme biosynthetic enzymes were examined in differentiating Neuro2a cells. During neuronal differentiation, there were increases in the cellular heme levels and increases in the mRNA levels for the rate-limiting enzymes of heme biosynthesis, such as aminolevulinic acid synthase (ALAS; EC 2.3.1.37) and coproporphyrinogen oxidase (EC 1.3.3.3). With respect to heme contents, heme b increased in the late phase of differentiation, but no apparent increase in heme a or b was observed in the early phase. In contrast, heme c (cytochrome c) markedly increased during the early phase of differentiation. This change preceded the increase in heme b and the up-regulation of the mRNA levels for heme biosynthetic enzymes. This study suggests the up-regulation of heme biosynthesis and differential regulation of the heme a, b, and c levels during neuronal differentiation.     

Carbon monoxide-binding cytochromes in the respiratory chain of the thermophilic bacterium PS3 grown with sufficient or limited aeration

The effects of aeration on the growth and cytochrome patterns of thermophilic bacterium PS3 were studied; bacteria grown with strong aeration synthesized cytochromes c, b, and aa3, while those grown with low aeration, showing non-exponential growth, synthesized higher amounts of cytochromes c and b including o, and a lower amount of cytochrome a (a3). The CO-difference spectra indicated that the terminal oxidase was cytochrome aa3 for high aeration conditions and the cytochrome o for low aeration conditions. Cytochrome o can be solubilized by Triton X-100 from the membrane fraction of bacteria grown under oxygen-limited conditions. The carbon monoxide complex of cytochrome o, obtained by exposing this extract to CO, was photolyzed and the subsequent rebinding of CO was analyzed; it followed first order kinetics with a rate constant of around 8 s-1 at 25 degrees C. At liquid nitrogen temperature, CO-rebinding did not occur. The CO-difference spectrum of purified cytochrome oxidase sample from the bacteria grown with strong aeration (Sone, N., et al. (1979) FEBS Lett. 106, 39-42) revealed the presence of a small amount of a cytochrome o-like pigment besides cytochrome aa3. Analysis of the CO complexes of these chromophores showed rate constants of 29-30 s-1 for cytochrome aa3 and 35-42 s-1 for the o-like pigment, indicating that the cytochrome o-like pigment contaminating the purified cytochrome oxidase preparation was not typical cytochrome o.     

Kinetics of electron transfer between cytochromes c' and the semiquinones of free flavin and clostridial flavodoxin

Rate constants have been measured for the reactions of a series of high-spin cytochromes c' and their low-spin homologues (cytochromes c-554 and c-556) with the semiquinones of free flavins and flavodoxin. These cytochromes are approximately 3 times more reactive with lumiflavin and riboflavin semiquinones than are the c-type cytochromes that are homologous to mitochondrial cytochrome c. We attribute this to the greater solvent exposure of the heme in the c'-type cytochromes. In marked contrast, the cytochromes c' are 3 orders of magnitude less reactive with flavodoxin semiquinone than are the c-type cytochromes. We interpret this result to be a consequence of the location of the exposed heme in cytochrome c' at the bottom of a deep groove in the surface of the protein, which is approximately 10-15 A deep and equally as wide. While free flavins are small enough to enter the groove, the flavin mononucleotide (FMN) prosthetic group of flavodoxin is apparently prevented by steric constraints from approaching the heme more closely than approximately 10 A without dynamic structural rearrangements. Most cytochromes c' are dimeric, but a few are monomeric. The three-dimensional structure of the Rhodospirillum molischianum cytochrome c' dimer suggests that the heme should be more exposed in the monomer than in the dimer, but no relationship is observed between intrinsic reactivity toward free flavin semiquinones and the aggregation state of the protein. Likewise, there is no evidence that the spin state or ligand field of the iron has any effect on intrinsic reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)