The Mitochondrial b=Cytochromes of the Wild Type and Poky Strains of Neurospora crassa

SUMMARY Mitochondria isolated from the wild type and poky strains of Neurospora crassa are shown to contain three b-cytochromes. Two of these-b-561 and b-566 (b-558)-are reduced by physiological substrates, whereas the third (CP peak (25”) at 558 nm) is reduced only by dithionite. The concentration of all three b-cytochromes is much lower in poky than in wild type mitochondria, but the ratio of b-561 to b-566 (b-558) remains 1: 1 in both strains. On the other hand, the proportion of “dithionite-reducible” b, measured by potentiometric titrations at wavelength pair 559-540 nm, increases from 30% in wild type to 55 to 60% in poky. The

are shown to contain three b-cytochromes.
Two of these-b-561 and b-566 (b-558)-are reduced by physiological substrates, whereas the third (CP peak (25") at 558 nm) is reduced only by dithionite.
The concentration of all three b-cytochromes is much lower in poky than in wild type mitochondria, but the ratio of b-561 to b-566 (b-558) remains 1: 1 in both strains. On the other hand, the proportion of "dithionite-reducible" b, measured by potentiometric titrations at wavelength pair 559-540 nm, increases from 30% in wild type to 55 to 60% in poky.
The presence of the dithionite-reducible component has been overlooked by other workers leading to an apparently erroneous report that the stoichiometry of b-561 and b-566 (b-558) is 6: 1 in poky mitochondria (VON JAGOW, G., WEISS, H., AND KLINGENBERG, M. (1973) Eur. J. Biochem. 33, 140). The measured midpoint potentials of b-561, b-566 (b-558), and dithionite-reducible b in wild type mitochondria are 78 f 2, -73 =L 8, and -166 f 4 mv, respectively. Roughly the same midpoint potentials are found for the b-cytochromes of poky and the results support the idea that, except for concentration, the b-cytochromes are not significantly altered by the poky mutation.
Poku (mi-1) is a cytoplasmically inherited mutant of ATeurospora crassa characterized by a slow growth rate, a defect in mitochondrial ribosomc assembly, and deficiencies in several cytochromes (l-4).
Most (about two-thirds) of poky's respiration is mediated by a novel hydroxamic acidsensitive osidase system which is resistant to the conventional respiratory inhibitors (cyanide and antimycin) and which is not present constitut,ively in wild type (5)(6)(7).
The remaining onethird of poky's respiration is mediated by the residual cytochrome system. The com-* This work was supported by grants from the National Science Foundation and the Herman Frasch Foundation.
$ To whom correspondence should be addressed at The Rockefeller University, New York, New York 10021. ponents of this system are qualitatively similar to those of wild type mitochondria, but with the exception of cytochrome c, they arc present at much lower concentrations (2)(3)(4)7). Even SO, since the alternate oxiclase is nonphosphorylating, the cytochrome system accounts for nearly all of the osidative phosphorylation in log phase poky cells (6). One matter of con troversy that has arisen regarding the poky cytochrome system concerns the number and stoichiomctry of the b-cytochromes. On t,he basis of studies by Lambowitz et al. (4) and von Jagow et al. (7), it is agreed that Neurospora mitochondria contain at least two b-cytochromes, b-561 and b-566 (b-558),', 2 aualogous to components found in animal and higher plant mitochondria, and that these two b-cytochromes are present. in equal conceutrat.ion iu the wild type strain judged from low temperature spectra of aerobic, antimycin-treated mitochondria reduced with succinate (conditions for which the b-cytochromes arc maximally reduced; see Fig. 1 of Ref. 4). For poky, on the other hand, von Jagow et al., estimating concentrations from spectra of dithionite-reduced mitochoutlria, reported that the concentration of b-561 was six times that of b-566 (b-558), while in the study of Lambowitz et al., with reduction by succiuatc plus antimyciu, it was estimated that the b-cytochromcs were present in equal concentrations (see Fig. 3 of Ref. 4). The result of van Jagow et a2. was purported to demonstrate that the stoichiometry of the b-cl complex is variable and might also suggest that incorporation of the two bcytochromes into the mitochondrial membraue is independently regulated.
However, the discrepancy is more prosaically explaiued by the possibility suggested by Lambowitz et al. that 1 Following the recommendation of t,he IUB Cytochrome Nomenclature Subcommittee, b-561 and b-566 are named according to the positions of their a-peaks in reduced minus oxidized difference spectra at room temperature.
In previous work on ATeurospora mitochondria, these components had been designated bsrr and b561 according to their 01 absorption maxima at 77K (Ref. 4) and bx and bT hy analogy with the nomenclature advanced by Chance and co-workers for animal mitochondria (7,20). 2 Room temperature difference spectra of cytochrome b-566 in mitochondria from animals (20), higher plants (II), yeast (22), and iYeurospora (7) show a minor a-peak at 558 nm, which is usually about one-half of the height of the main peak at 566 nm. Whether the 558.nm peak belongs to b-566 or whether it is a separate low potential component is uncertain and will remain so until b-566 has been highly purified.
In the present manuscript, for ease of presentation, we have adopted the nomenclature The present paper suggests that the latter is in fact the case and provides spectrophotometric and potentiometric evidence for the presence of a "dithionite-reducible" cytochrome b in both the wild type and poky strains of N. crassa.

MATERIALS AND METHODS
Strains of Neurospora-The wild type strain, RL2la, was derived from the original Linderrren wild tvnes LlA and L25a (8). The poky strains, NEX f+ a andNS8 f-A &&e obtained from &. D. Luck, The Rockefeller University. f+ is a nuclear gene suppressor of the poky mutant, increasing the growth rate of strains which carry it without restoring the wild type respiratory system (5,9).
Growth of Cells-Cells were grown in aerated liquid cultures at 25" in Vogel's minimal medium (10) plus 2% sucrose and were harvested during late exponential phase: 15% hours for wild type, 20 to 21 hours for poky f+, and 23 to 24 hours for poky f-. Procedures for maintaining strains and preparing conidia were as described by Slayman and Tatum (8).
Reagents-Reagents were of the highest grade available. Antimycin (Calbiochem) and bis-(hexafluoroacetonyl)acetone 1799 (obtained from Dr. P. Heytler, E. I. duPont de Nemours Co., Wilmington, Del.) were dissolved in absolute ethanol. NADH (Boehringer Mannheim) was dissolved in 0.05 M sodium carbonate. All other reagents were dissolved in distilled water and adjusted to pH 7. Preparation of oxidation-reduction mediator solutions has been described previously (11).
Preparation of Mitochonclria-Mitochondria were prepared by osmotic lysis of snail enzyme-treated cells followed by two cycles of differential centrifugation (6). The mitochondrial preparations were virtually free of microsomal contamination as judged by the recovery of more than 95% of the mitochondrial protein in a single band following isopycnic sedimentation on discontinuous Sucrose gradients if the type used by Deuce et al. for the nurification of higher nlant mitochondria (12). Mitochondrial protein was determined Gy the method of Lowry et al. (13) using crystalline bovine serum albumin (Miles Laboratories) as a standard.
Spectrophotometric Measurements-Spectra of cytochromes at room temperature and at 77 K were obtained with a scanning split beam spectrophotometer (14). The time course and extent of oxidation and reduction of the cytochromes were measured with a dual wavelength spectrophotometer (14). For purposes of computation in the spectrophotometric experiments, the cytochromes were assumed to be fully oxidized in aerobic mitochondria lacking substrate.
The extinction coefficient of cytochrome b was assumed to be 20 rnM-1 cm-l (15).
Potentiometric Titrafions-Potentiometric titrations were carried out by the method of Dutton et al. (16) making use of a dual wavelength spectrophotometer in combination with an appropriate electrode system.

Spectrophotometric
Resolution of Dithionite-reducible Cytochrome b-The idea that Neurospora mitochondria contain a blike component reduced only by dithionite was originally based on the observation that total cytochrome b was only about 50% reduced upon anaerobiosis with succinate or NADH (4). Proceeding from this result, it is necessary to distinguish between dithionite-reducible b and b-566 (b-558), since the latter cytochrome has a low midpoint potential and is also known to be incompletely reduced by physiological substrates (4,7,17). The approach adopted was to force reduction of b-566 (b-558) by taking advantage of the large increase in its reducibility which can be induced by antimycin plus oxidant (4,7,17,18). The mechanism of the antimycin effect is unknown, but the dithionite-reducible b does remain oxidized under these conditions so that the effect can be used to resolve the two components. Fig.  1 shows spectra for wild type and poky f+ obtained with this approach by using the dual wavelength spectrophotometer and plotting the difference between the oxidation-reduction level in the presence of dithionite and that in the presence of succinate plus antimycin as a function of measuring wavelength.
In the cr region, the spectra show a single peak, at 558 or 559 nm, equivalent to 0.2 nmole of cytochrome b per mg of protein in wild type and about one-fourth that amount (0.05 nmole of cytochrome b per mg of protein) in poky f+. For both strains, the dithionite-reducible component contributes at least 30 to 50% of the total b-cytochrome absorbance at wavelengths between 558 and 560 nm and the lower absolute concentration in poky merely reflects the deficiency of total cytochrome b in this strain. Fig. 2 shows analogous spectra recorded at 77 K using the split beam spectrophotometer.
The spectral peaks, shifted a few nanometers to the blue at low temperature, remain characteristic of a b type cytochrome (a-peak at 556 nm, P-peaks 527 to 535 nm, and Soret peak at 424 nm). In addition, with the increased resolution at low temperature (19), distinct shoulders are seen associated with the u-peaks in both strains (551 and 562 nm for wild type and 552 nm for poky f+). It is not known whether these shoulders are part of the dithionite-reducible b or whether they are due to a small amount of b-566 (b-558) that remains oxidized in the reference cuvette.
The peaks at 545 to 547 nm are presumably due to interference from cytochrome c.
Potentiometric Titrations-The spectrum of the dithionitereducible cytochrome b is similar to that of b-561. Indeed, thus far the main distinction is that the latter is assumed to be completely reduced upon anaerobiosis in the presence of succinate, whereas the former remains oxidized under these conditions. The reference sample was treated with 10 mM show complete titration data, whereas those to the right show succinate (plus 0.1 rnM N,N,N',N'-tetramethyl-p-phenylene-resolution of components using the arithmetic method of Dutton diamine dihydrochloride in the case of wild type) and, after et al. (16). Details of the experimental procedure are given in anaerobiosis, antimycin was added at a concentration of 10 pg Refs. 11, 16, and 23. The mitochondria were suspended in stanper ml. At the same time, antimycin was added to the measuring dard reaction medium (pH 7.2; see "Materials and Methods") cuvette to eliminate the possibility of interference from anti-and uncoupled by 2-min soak in ATP (0.17 mM) followed by addimycin-induced spectral shifts. The reference sample was then tion of 1799 (3.3 PM). Titrations were carried out reductively vigorously pipetted to induce a transient aerobic state, ensuring (i.e. by slowly lowering the oxidation-reduction potential). The maximal reduction of (and minimal interference from) b-566 following oxidation-reduction mediators were present: diamino- In that case, what we have desigfigure are considered to be b-561, b-566 (b-558), and the dithionitenated dithionite-reducible cytochome b would simply be the reducible cytochrome b, respectively. oxidized portion of b-561 rather than a separate component.
In order to eliminate this uncertainty and also to obtain a better  (16). This Potentiometric titrations were carried out as in Fig. 3 and E, technique has had wide application in resolving cytochromes values are mean f S.E.M. for three determinations. Numbers in which have overlapping spectra but which differ in midpoint parentheses are relative contributions of components expressed potential (11,16,(22)(23)(24). Fig. 3 shows representative titrations as per cent of the total b-cytochrome absorbance change (559-540 for wild type and poky f+ mitochondria at wavelength pair 559nm)' 540 nm. Indeed the data for both strains do fall on a curve E, values showing three major components indicative of at least three b-cytochromes.
The plots are resolved to the right in Fig. 3 cytochromes. Table I (7). Similarly, from the spectrophotometric studies (see above and Refs. 4 2ss9 predominant low potential component at the ~vavelrngth pair used in the titrations, and also that there should be a smaller contribution from b-566 (b-558). For all three cytochromes the E, values are in close agreement rvith those of the analogous b components in animal, yeast, and higher plant mitochondria (11,16,22,23).
An outstanding feature of the potentiomctric titrations is that they permit accurate determination of the relative contributions of the different b components.
Significantly, the data in Table I show that the proportion of dithionite-reducible b is about twice as great in poky f+ as in tvild type mitochondria (55 and 3Oc& respectively, at the Ivavelength pair used) \vhile, 011 the other hand, the ratio of b-561 to b-566 (b-558) is about the same in the two types of mitochondria.
In subscqutnt cspcriments, results similar to those for poky ff Ivere obtained for pokg fP \vith the dithionitc-reducible b contributing about 60';; of the total b absorbance and \I-ith no change in the ratio of b-561 to b-566 (b-558).
The data presented in this report sho\~ that mitochondria from both the Iviltl type and poky strains of N. crassa contain at least three b cytochromes: b-561, by566 (b-558), and a dithionitereducible b. b-561 and b-566 (b-558) are apparently analogous to components found in animal and plant mitochondria (11, a&22) and sometimes referred to as bK and bT, respectively (20). Some properties of these components in ,Veurospora mitochondria have been discussed previously (4,7). A dithionite-reducible component has also been reported for SOIIIP other tgpcs of mitochondria, including beef heart (16,25), yeast (22), and higher plants (ll), and in each case, the measured E,, has been found to be about --100 mv (11,16,22). For .Veurospora and higher plants, there is good evidence that the dithionite-reducible b is truly a mitochondrial component (and not a microsomal contaminant) since it is present in very pure mitochondrial preparations. The Neurospora mitochondria used in the present rvork are judged to be free of microsomal contamination by isopycnic sedimentation (see "Uaterials and ;\Iethods"), and it should be added that the gradient-purified mitochondria ivcrc tested and found to be identical to those ordinarily used Gth respect to bot,h the spectrum and concentration of tlithionite-reducible b.
The fmlction, if any, of the dithionite-reducible b is not kno\vn. The component is considered to be a b-cytochrome 011 the basis of its characteristic b-cytochrome absorption spectrum (Fig. 2) and its ability to function as a l-electron carrier in the potentiometric titrations (Fig. 3). However, the term "cytochrome" also implies that the component function physiologically by shuttling betxeen the Fe2+ and Fe3f states and this has not been demon strated in any system. It should be noted that some types of animal mitochondria (e.g., pigeon heart mitochondria) apparently do not have a dithionite-reducible b (16,20), a fact which indicates that the component cannot be an essential part of the respiratory chain. Indeed, the relatively high concentration of the dithionite-reducible b in poky, which is known to accumulate precursor and nonfunctional forms of other respiratory chain components (26,27), lends support to the view that it is nascent or damaged cytochrome b. In this regard, it is interesting to note the recent observation of Sato et al. (28) that a component with properties similar to dithionite-reducible b increases in concentration at the expense of the respiratory chain b-cytochromes in L cells treated with ethidium bromide (a substance lvhich blocks formation of functional electron transport systems).
As expected, the concentration of all three b-cytochromes is lolvcr in poky than in Iviltl type mitochondria. Holvevcr, it can be concluded (a) that the ratio of b-561 and b-566 (b-558) remains 1 :l in both strains (from spectra of antimycin-treated mitochondria reduced \\-ith succinate (4), ivith support from the potentiometric titrations reported here), and (b) that the dithionite-reducible b is present in more variable concentrations, comprising a greater proportion of the total b in poky than in wild type. The presence of a separate dithionite-reducible component, although suggested previously (4), leas overlooked by von Jagow ef al. (7) and this oversight probably accounts for the unusually high ratio of b-561 to b-566 (b-558) \vhich they reported for poky mitochondria.
Indeed, it should be added that, apart, from interpretation, the data in the van Jagolv study agree very lvell \vith the concept, of a dithionite-reducible component since their spectra also indicate incomplete reduction of cytochrome b by succinate.
It is possible, honevcr, that their \vild type mitochondria contained somelvhat less, and their poky mitochondria somelvhat more, dithionite-reducible b than those used in the present work.
The measured IG', values of the three 6 spccirs arc summarized in Table I. hs noted in an earlier study, the lcrn values measured by the techniques llow available may bc less accurate than the precision of the measurements suggests (11). Holvever, the data are sufficient to show that there is no gross difference in the E, values between the Gltl type and poky f+ strains. This result is in agreement with the earlier generalization that the same b components are present in both strains and that, cscept ivith respect to concentration, they are not significantly altered by the poky mutation (4).
The sequence of electron transport reactions in the b region of h'eurospora is an important problem \vhich must no\v be approached by kin&c experiments.
The contention of von Jagow et al. (7) that the sequence is b-561 to b-566 (b-558) with the latter cytochrome located directly on the substrate side of "site II" is premature. l\Iorcover, this contention \vas based primarily on experiments nith animal mitochondria purporting to sholv "heme~heme interaction" betlvecn b-566 (b-558) and cytochrome c1 and participation of b-566 (b-558) in energy transduction at site II (29-31).
Virtually all of this evidence has since been brought into question (32-37, 11, 17). At present, the only evidence bearing on the ordering question in h'eurospora mitochondria is in fact the potcntiometric data in this report and these data suggest that b-561 (the high potential species) is on the osygen side of b-566 (b-558).