Mitochondrial assembly in respiration-deficient mutants of Saccharomyces cerevisiae. IV. Effects of nuclear amber suppressors on the accumulation of a mitochondrially made subunit of cytochrome c oxidase.

Earlier studies from this laboratory have shown that cytochrome c oxidase from bakers' yeast contains seven subunits, three of which are made in the mitochondrion (Mason, T. L., and Schatz, G. (1973) J. Biol. Chem. 248, 1355). Moreover, a cytochrome c oxidase-less yeast mutant (pet 494-1) was isolated which lacked one of the mitochondrially made subunits (Ebner, E., Mason, T. L., and Schatz, G. (1973) J. Biol. Chem. 248, 5369). Surprisingly, the mutated gene was localized in the nucleus. The results presented here demonstrate that this mutant phenotype can be suppressed by nuclear amber suppressors which affect translation on cytoplasmic ribosomes. This fact was established by two methods, (a) By constructing pet 494-1 strains possessing various amber and ochre markers, isolating respiring revertants from these strains, and demonstrating co-reversion of the amber (but not of the ochre) markers. (b) By coupling the pet 494-1 allele with the well characterized amber suppressor gene SUP 4-3. These data show that suppressor genes located on nuclear chromosomes may control the accumulation of a mitochondrially synthesized polypeptide. The present results also allow some tentative conclusions about the mechanism of the pet 494 mutation. Because it is highly unlikely that the cytoplasmic and the mitochondrial translation system share a common suppressor, the pet 494 locus probably does not code for the missing mitochondrially made subunit, but for a cytoplasmically made protein. This as yet unidentified protein seems to control the synthesis or the integration of the mitochondrially made subunit. Nuclear suppressor genes may thus be useful tools for studying the role of cytoplasmic protein synthesis in mitochondrial formation.     

Cytochrome c oxidase from bakers' yeast. Photolabeling of subunits exposed to the lipid bilayer.

Yeast mitochondria and purified yeast cytochrome c oxidase incorporated into micelles of the nonionic detergent Tween 80 were equilibrated with the hydrophobic aryl azides 5-[125I]iodonaphthyl-1-azide or S-(4-azido-2-nitrophenyl)-[35S]thiophenol. The azides were then converted to highly reactive nitrenes by flash photolysis or by illumination for 2 min and the derivatized cytochrome c oxidase subunits were identified by gel electrophoresis and radioactivity measurements. 5-[125I]Iodonaphthyl-1-azide labeled mainly the three mitochondrially made Subunits I to III and the cytoplasmically made Subunit VII. Subunits IV to VI or cytochrome c bound to the purified enzyme were labeled 9- to 90-fold less. Essentially the same result was obtained with S-(4-azido-2-nitrophenyl)-[35S]thiophenol except that Subunit V was labeled as well. In contrast, all seven subunits as well as cytochrome c were heavily labeled when the enzyme was dissociated with dodecyl sulfate prior to photolabeling with either of the two probes. These data indicate that all subunits of yeast cytochrome c oxidase except Subunits IV and VI are at least partly embedded in the lipid bilayer of the mitochondrial inner membrane.     

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.     

Heme is necessary for the accumulation and assembly of cytochrome c oxidase subunits in Saccharomyces cerevisiae.

The presence of cytochrome c oxidase subunits and the association of these subunits with each other was studied in a heme-deficient Saccharomyces cerevisiae mutant. This mutant had been isolated by Gollub et al. (1977) J. Biol. Chem. 252, 2846-2854) and had been shown lack delta-aminolevulinic acid synthetase. When grown in the absence of heme or heme precursors, the mutant is respiration-deficient, devoid of cytochrome absorption bands and auxotrophic for all those components whose biosynthesis is dependent on hemoproteins; when grown in the presence of heme or heme precursors, the mutant is phenotypically wild type. Upon growth of the mutant in the absence of heme synthesis, the mitochondria still contained two of the three mitochondrially made cytochrome c oxidase subunits (i.e. II and III) and at least one of the cytoplasmically made cytochrome c subunits (VI). The other subunits were either barely detectable (I, IV) or undetectable (V, VII). The residual subunits were apparently not assembled with each other since an antiserum directed mainly against Subunit VI failed to co-precipitate Subunits II and III which were still present. In contrast, growth of the mutant in the presence of delta-aminolevulinic acid led to the accumulation of active, fully assembled cytochrome c oxidase in the mitochondria. Heme a (or one of its precursors) thus controls the assembly of cytochrome c oxidase from its individual subunits.     

Cytochrome c oxidase from bakers' yeast. IV. Immunological evidence for the participation of a mitochondrially synthesized subunit in enzymatic activity.

In order to study the role of the individual subunits of yeast cytochrome c oxidase, rabbit antisera were prepared against Subunit II (a mitochondrially made polypeptide) and Subunit VI (a cytoplasmically made polypeptide). Antisera were also obtained against a mixture of the two mitochondrially made subunits (I PLUS II) and against mixtures of the following cytoplasmically made subunits: (IV PLUS VI); (V PLUS VII); and (IV PLUS V PLUS VI PLUS VII). Neither anti-II serum nor anti-VI serum cross-reacted with any of the other six subunits of cytochrome c oxidase as judged by a sensitive ring test or by double diffusion in agarose gels. Anti-II serum inhibited the oxidation of ferrocytochrome c by purified yeast cytochrome c oxidase or by freshly isolated as well as sonically fragmented yeast mitochondria. Anti-(V, VII) serum and anti-(IV, V, VI, VII) serum were also strongly inhibitory. Anti-VI serum and anti-(IV, VI) serum inhibited only weakly. If purified cytochrome c oxidase was inhibited with a saturating amount of anti-VI serum, anti-II serum elicited a further increment of inhibition, as would be expected if the inhibitory effects of these two antisera involved different antigenic sites on the holoenzyme. Each of the antisera precipitated all seven cytochrome c oxidase subunits from crude mitochondrial extracts. However, anti-VI and, particularly, anti-II were much less effective precipitants than antisera against Subunits IV to VII or antisera against the holoenzyme. These data suggest that the oxidation of ferrocytochrome c by cytochrome c oxidase required both mitochondrially as well as cytoplasmically made subunits.     

Mitochondrial gene expression in saccharomyces cerevisiae. I. Optimal conditions for protein synthesis in isolated mitochondria

An in vitro mitochondrial protein-synthesizing system, which makes use of intact yeast mitochondria, has been developed in order to study mitochondrial gene expression and its control by nuclear-coded proteins. Studies with this system have revealed that: isolated mitochondria synthesize polypeptide gene products which can be radiolabeled to high specific radioactivities when incubated in a "protein-synthesizing medium" that has been optimized with respect to each of its components; two energy-generating systems, endogenous oxidative phosphorylation and an exogenous ATP-regenerating system, support the highest level of protein synthesis; and the omission of an oxidizable substrate results in the synthesis of two new polypeptides (19.5 and 18 kDa) and a decrease in the amounts of cytochrome c oxidase subunits I and II which are synthesized. They have also revealed that added adenine and guanine nucleotides increase the overall level of protein synthesis and that the added guanine nucleotides facilitate polypeptide chain elongation. Although isolated mitochondria which have been optimized for protein synthesis synthesize normal gene products (McKee, E. E., McEwen, J. E., and Poyton, R. O., (1984) J. Biol. Chem. 259, 9332-9338) they still respond to an added dialyzed S-100 fraction from yeast cells by increasing their level of protein synthesis. This stimulation is observed in the presence of optimal concentrations of GTP, making it unlikely that guanyl nucleotides or enzymes which synthesize them are the sole stimulatory factors present in cellular cytosolic fractions, as suggested by Ohashi and Schatz (Ohashi, A., and Schatz, G. (1980) J. Biol. Chem. 255, 7740-7745).     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Identification of cytochrome c oxidase subunits in nuclear yeast mutants lacking the functional enzyme.

Yeast mutants specifically lacking cytochrome c oxidase activity were screened for cytochrome c oxidase subunits by one- and two-dimensional electrophoresis, electrophoresis in exponential gradient gels, and immunoprecipitation with antisera against one or more of the cytoplasmically made subunits of the enzyme. Two cytochrome c oxidase-less nuclear mutants previously described from this laboratory each lack one or more mitochondrially synthesized cytochrome c oxidase subunits while possessing all four cytoplasmically synthesized subunits of that enzyme. The subunits remaining in these mutants were not assembled with each other; the cytoplasmically made subunits IV and VI could be released from the mitochondria by sonic oscillation, in contrast to the situation in wild type cells. No electrophoretically detectable alterations were found in any of the cytochrome c oxidase subunits present in the mutants. Nuclear mutations may thus cause both a loss as well as a defective assembly of mitochondrially made cytochrome c oxidase subunits.     

COX8, the structural gene for yeast cytochrome c oxidase subunit VIII. DNA sequence and gene disruption indicate that subunit VIII is required for maximal levels of cellular respiration and is derived from a precursor which is extended at both its NH2 and COOH termini

From the amino acid sequence of yeast cytochrome c oxidase subunit VIII published previously (Power, S. D., Lochrie, M.A., Patterson, T.E., and Poyton, R.C. (1984) J. Biol. Chem. 259, 6571-6574), we have synthesized a pair of oligonucleotide probes and used them to identify COX8, its structural gene. By genomic Southern blot analysis and disruption of the COX8 chromosomal locus, we have shown that this gene is present in one copy per haploid genome and that its product, subunit VIII, is essential for maximal levels of cellular respiration and cytochrome c oxidase activity. Alignment of the amino acid sequence predicted from the DNA sequence of COX8 with the determined amino acid sequence of subunit VIII indicates that mature subunit VIII is derived from a larger precursor that extends from both the NH2 and COOH termini of the mature polypeptide. Thus, like many other nuclear coded mitochondrial proteins, subunit VIII is derived from a precursor which carries a leader peptide. In addition, this precursor, like that for yeast cytochrome c oxidase subunit VIIa, appears to carry a four-amino acid "trailer peptide" at its COOH terminus.     

ATP induces conformational changes in mitochondrial cytochrome c oxidase. Effect on the cytochrome c binding site

ATP influences the kinetics of electron transfer from cytochrome c to mitochondrial oxidase both in the membrane-embedded and detergent-solubilized forms of the enzyme. The most relevant effect is on the so-called "high affinity" binding site for cytochrome c which can be converted to "low affinity" by millimolar concentrations of ATP (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115). This phenomenon is characterized at the molecular level by the following features. ATP triggers a conformational change on the water-exposed surface of cytochrome c oxidase; in this process, carboxyl groups forming the cluster of negative charges responsible for binding cytochrome c change their accessibility to water-soluble protein modifier reagents; as a consequence the electrostatic field that controls the enzyme-substrate interaction is altered and cytochrome c appears to bind differently to oxidase; photolabeling experiments with the enzyme from bovine heart and other eukaryotic sources show that ATP cross-links specifically to the cytoplasmic subunits IV and VIII. Taken together, these data indicate that ATP can, at physiological concentration, bind to cytochrome c oxidase and induce an allosteric conformational change, thus affecting the interaction of the enzyme with cytochrome c. These findings raise the possibility that the oxidase activity may be influenced by the cell environment via cytoplasmic subunit-mediated interactions.     

Yeast cytochrome c oxidase subunit VII is essential for assembly of an active enzyme. Cloning, sequencing, and characterization of the nuclear-encoded gene

The gene COX VII coding for yeast cytochrome c oxidase subunit VII has been cloned by a two-step procedure. Two degenerate oligonucleotides corresponding to amino- and carboxyl-terminal protein segments were used in a polymerase chain reaction for the amplification of a major portion of subunit VII (residues 1-52), which was then used for the cloning of complete COX VII. From the nucleotide sequence, an additional amino-terminal and two additional carboxyl-terminal amino acids are predicted as compared with the described primary sequence (Power, S. D., Lochrie, M. A., and Poyton, R. O. (1986) J. Biol. Chem. 261, 9206-9209). Beside subunit VIIa the subunit described here is the only nuclear encoded subunit of cytochrome c oxidase in yeast without a leader sequence. COX VII exists as a single copy per haploid genome as shown by Southern blot and gene disruption. Null mutants produced by gene disruption at the COX VII locus were respiratory-deficient. No cytochrome c oxidase activity was detectable nor was there an assembly of the oxidase complex.     

Cytochrome c oxidase subunits in contact with phospholipids. Hydrophobic photolabeling with azidophospholipids.

The technique of photolabeling of membrane proteins with arylazidophospholipids was applied to cytochrome c oxidase. The "deep" and "shallow" labels employed reacted with all subunits of cytochrome c oxidase except V and VI: Subunits I, III, and VII were heavily labeled, Subunit II was labeled to a lesser extent, and Subunit IV was poorly labeled. Subunit I was labeled more by the deep label and Subunit VII by the shallow one. The other subunits were equally labeled by the two probes. This technique has revealed what subunits of cytochrome c oxidase interact with the lipid and their approximate position in the membrane.     

Cytochrome c oxidase: structure, function, and membrane topology of the polypeptide subunits.

Mitochondrial cytochrome c oxidase and its bacterial homologs catalyze electron transfer and proton translocation reactions across membranes. The eukaryotic enzyme complex consists of a large number of polypeptide subunits. Three of the subunits (I, II, and III) are mitochondrially encoded while the remaining 6 (yeast) to 10 (bovine) are nuclear encoded. Antibody and chemical-labelling experiments suggest that subunits I-III and most (but not all) of the nuclear-encoded subunits span the inner mitochondrial membrane. Subunits I and II are the catalytic core of the enzyme. Subunit I contains haem a, haem a3 and CuB, while subunit II contains CuA and the cytochrome c binding site. Subunit III and most of the nuclear subunits are essential for the assembly of a functional catalytic enzyme. Some nuclear subunits are present as isozymes, although little functional difference has yet been detected between enzyme complexes composed of different isozymes. Therefore, any additional role attributed to the nuclear-encoded subunits beyond that of enzyme assembly must be tentative. We suggest that enough evidence exists to support the idea that modification of the larger nuclear subunits (IV, V, and possibly VI) can effect enzyme turnover in vitro. Whether this is a physiological control mechanism remains to be seen.     

Cytochrome c binding affects the conformation of cytochrome a in cytochrome c oxidase.

Second derivative absorption spectroscopy has been used to assess the effects of complex formation between cytochrome c and cytochrome c oxidase on the conformation of the cytochrome a cofactor. When ferrocytochrome c is complexed to the cyanide-inhibited reduced or mixed valence enzyme, the conformation of ferrocytochrome a is affected. The second derivative spectrum of these enzyme forms displays two electronic transitions at 443 and 451 nm before complex formation, but only the 443-nm transition after cytochrome c is bound. This effect is not induced by poly-L-lysine, a homopolypeptide which is known to bind to the cytochrome c binding domain of cytochrome c oxidase. The effect is limited to cyanide-inhibited forms of the enzyme; no effect was observed for the fully reduced unliganded or fully reduced carbon monoxide-inhibited enzyme. The spectral signatures of these changes and the fact that they are exclusively associated with the cyanide-inhibited enzyme are both reminiscent of the effects of low pH on the conformation of cytochrome a (Ishibe, N., Lynch, S., and Copeland, R. A. (1991) J. Biol. Chem. 266, 23916-23920). These results are discussed in terms of possible mechanisms of communication between the cytochrome c binding site, cytochrome a, and the oxygen binding site within the cytochrome c oxidase molecule.     

A role for membrane potential in the biogenesis of cytochrome c oxidase subunit II, a mitochondrial gene product

Subunit II of yeast cytochrome c oxidase is synthesized on mitochondrial ribosomes as a precursor containing a transient NH2-terminal presequence and is inserted into the mitochondrial inner membrane from the matrix side. Using an optimized in vitro mitochondrial translation system (McKee, E.E., and Poyton, R. O. (1984) J. Biol. Chem. 259, 9320-9331), we have examined the requirement for an electrochemical potential (delta mu H+) across the inner mitochondrial membrane during subunit II biogenesis. When mitochondrial gene products are synthesized under conditions that prevent formation of a normal delta mu H+, accumulation of unprocessed subunit II (pre-II) occurs. The majority of pre-II generated in this way is inserted into the lipid bilayer, as judged by resistance to extraction with 0.1 M Na2CO3. Therefore, it appears that a delta mu H+ is required for the normal biogenesis of subunit II, and that the delta mu H+ is required for a function other than the insertion of pre-II into the lipid bilayer of the inner membrane.     

Characterization of electron-transfer and proton-translocation activities in bovine heart mitochondrial cytochrome c oxidase deficient in subunit III

The electron-transfer and proton-translocation activities of cytochrome c oxidase deficient in subunit III (Mr 29 884) prepared by native gel electrophoresis [Ludwig, B., Downer, N. W., & Capaldi, R. A. (1979) Biochemistry 18, 1401-1407] have been investigated. This preparation has been depleted of 82-87% of its subunit III content as quantitated by Coomassie Brilliant Blue staining intensity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and [14C]dicyclohexylcarbodiimide labeling. The maximum rate of electron transfer of the subunit III deficient enzyme at pH 6.5 is 383 s-1, 78% of control enzyme. Neither the high-affinity site (Km = 10(-8) M) nor the low-affinity site (Km = 10(-6) M) of the cytochrome c kinetic interaction with cytochrome c oxidase is affected by the removal of subunit III. Subunit III deficient cytochrome c oxidase retains the ability to bind cytochrome c in both the high- and low-affinity sites as determined in direct thermodynamic binding experiments. Liposomes containing this preparation exhibit a respiratory control ratio [Hinkle, P. C., Kim, J. J., & Racker, E. (1972) J. Biol. Chem. 247, 1338-1341] of 3.9, while liposomes containing control enzyme exhibit a ratio of 4.3, suggesting that they have a similar proton permeability. Vectorial proton translocation initiated by the addition of ferrocytochrome c in liposomes containing subunit III deficient enzyme is decreased by 64% compared to those containing control enzyme. When the proton-translocated to electron-transferred ratio is measured in these phospholipid vesicles at constant enzyme turnover, removal of subunit III from the enzyme decreases the ratio from 0.52 to 0.21, a 60% decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen influences the subunit structure of cytochrome c oxidase in the slime mold Dictyostelium discoideum

The conditions that promote the alternative expression of two nuclear-encoded subunits of cytochrome c oxidase in the slime mold Dictyostelium discoideum (Bisson, R., and Schiavo, G. (1986) J. Biol. Chem. 261, 4373-4376) have been investigated. Oxygen concentration seems to be the only factor able to cause the subunit switching. This result indicates that the polypeptide composition of the mitochondrial enzyme can be influenced by environmental conditions. The significance of this change is discussed.     

Structure of an electron transfer complex. II. Chemical modification of carboxyl groups of cytochrome c peroxidase in presence and absence of cytochrome c

Cytochrome c peroxidase forms an electron transfer complex with cytochrome c. The complex is governed by ionic bonds between side chain amino groups of cytochrome c and carboxyl groups of peroxidase. To localize the binding site for cytochrome c on the peroxidase, we have used the method of differential chemical modification. By this method the chemical reactivity of carboxyl groups (toward carbodiimide/aminoethane sulfonate) was compared in free and in complexed peroxidase. When ferricytochrome c was bound to cytochrome c peroxidase, acidic residues 33, 34, 35, 37, 221, 224, and 1 to 3 carboxyls at the C terminus became less reactive by a factor of approximately 4, relative to the remaining 39 carboxylates of peroxidase. Of the less reactive residues those in the 30-40 region and the 221/224 pair are on opposite sides of the surface area which contains the heme propionates. We, therefore, propose that the binding site for cytochrome c on cytochrome c peroxidase spans the area where one heme edge comes close to the molecular surface. The results are in very good agreement with chemical cross-linking studies (Waldmeyer, B., and Bosshard, H.R. (1985) J. Biol. Chem. 260, 5184-5190); they also support a hypothetical model predicted on the basis of the known crystal structures of cytochrome c and peroxidase (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330).     

The role of oxygen in the biosynthesis of cytochrome c oxidase of yeast mitochondria.

The formation of cytochrome c oxidase in yeast is dependent on oxygen. In order to examine the oxygen-dependent formation of the active enzyme, the effect of oxygen on the synthesis and the assembly of cytochrome c oxidase subunits was studied. Pulse-labeling experiments revealed that oxygen has no significant immediate effect on the synthesis of the three mitochondrially made subunits I to III; however, its presence causes subunits I and II to form a complex with the cytoplasmically made subunits VI and VII. This "assembly-inducing" effect can be demonstrated with intact yeast cells as well as with isolated mitochondria. It is independent of cytoplasmic or mitochondrial protein synthesis. After anaerobic growth for 10 or more generations, the intracellular concentrations of individual cytochrome c oxidase subunits drop 10- to 100-fold. Most of these residual subunits are not assembled within a functional cytochrome c oxidase molecule.     

Sulfite oxidase from chicken liver. The role of imidazole and carboxyl groups for the reaction with cytochrome c

Oxidation of sulfite to sulfate by sulfite oxidase is inhibited when the enzyme is treated with reagents known to modify imidazole and carboxyl groups. Modification inhibits the oxidation of sulfite by the physiological electron acceptor cytochrome c, but not by the artificial acceptor ferricyanide. This indicates interference with reaction steps that follow the oxidation of sulfite by the enzyme's molybdenum cofactor. Reaction with diethylpyrocarbonate modifies ten histidines per enzyme monomer. Loss of activity is concomitant to the modification of only a single histidine residue. Inactivation takes place at the same rate in free sulfite oxidase and in the sulfite-oxidase--cytochrome-c complex. Blocking of carboxyl groups with water-soluble carbodiimides inactivates the enzyme. But none of the enzyme's carboxyl groups seems to be essential in the sense that its modification fully abolishes activity. The pattern of inactivation by chemical modification of sulfite oxidase is quite similar to that observed previously for cytochrome c peroxidase from yeast [Bosshard, H. R., B?nziger, J., Hasler, T. and Poulos, T. L. (1984) J. Biol. Chem. 259, 5683-5690; Bechtold, R. and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5191-5200]. The two enzymes have very different structures yet share cytochrome c as a common substrate of which they recognize the same electron-transfer domain around the exposed heme edge.