Definition of cytochrome c binding domains by chemical modification. Interaction of horse cytochrome c with beef sulfite oxidase and analysis of steady state kinetics.

Enzymic conversion of inorganic sulfite to sulfate is the terminal step in sulfur catabolism in higher animals (1-6) and has also been observed in plants and bacteria (7-11). The physiological significance of this reaction is apparent from the severe pathophysiological consequences of its absence (6, 12, 13). Sulfite oxidase has been extensively purified (1-4, 14, 15) and

crosses the surface of the protein, indicating that the primary interaction of cytochrome c with its oxidationreduction partners is directed by the asymmetric charge distribution on the protein.
Enzymic conversion of inorganic sulfite to sulfate is the terminal step in sulfur catabolism in higher animals (1-6) and has also been observed in plants and bacteria (7-11). The physiological significance of this reaction is apparent from the severe pathophysiological consequences of its absence (6,12,13). Sulfite oxidase has been extensively purified (1-4, 14, 15) and is one of only a small number of enzymes known to contain molybdenum (16)(17)(18)(19)(20)(21). As with other molybdoenzymes a second prosthetic group is present, in this case a bs-type heme (21). The molybdenum and the heme exist in discrete domains contained in a 57,000 molecular weight polypeptide and are easily separated following limited tryptic digestion of the protein (22). The holoenzyme is a water-soluble dimer, located in the intermembrane space of mitochondria (23) where it has been shown to reduce cytochrome c (24). Electrons from sulfite reduce the molybdenum and are transferred to the b5 heme which is the electron donor for cytochrome c (21,25).
The steady state reduction of ferricytochrome c by sulfite oxidase has been investigated under a variety of conditions (23). Initial studies by Howell and Fridovich demonstrated that the effect on the enzymic reaction of varying the concentration of sulfite and cytochrome c is consistent with a pingpong mechanism (4). However, Kessler and Rajagopalan (26) showed that inhibition of the sulfite oxidase reaction by inorganic anions was not always competitive with respect to sulfite and cytochrome c. Small monovalent anions were found to be mixed noncompetitive inhibitors of the reaction of sulfite with sulfite oxidase, and competitive inhibitors of the reaction of cytochrome c with sulfite oxidase. Larger monovalent anions and multivalent anions are mixed noncompetitive inhibitors of the reaction of both sulfite and cytochrome c with the enzyme. This is consistent with distinct sites for the interaction of sulfite and cytochrome c and can be described as a hybrid ping-pong mechanism (27).
In order to understand the mode of electron transfer between cytochrome c and its mitochondrial oxidation-reduction partners, it is necessary to determine the area (or areas) on the surface of the molecule involved in the interaction and, therefore, presumably in the electron transfer process. The interaction domains on the surface of cytochrome c have been determined for beef cytochrome c oxidase and reductase, as well as yeast cytochrome c peroxidase, using a variety of singly These domains have also been mapped by determining the relative chemical reactivity of r-amino groups of lysines on cytochrome c free in solution as compared to those on cytochrome c complexed to purified preparations of its protein oxidation-reduction partners (37-39). At neutral pH, approximately 34 amino acid residues on horse cytochrome c are ionized, and since the resulting charges are strongly asymmetrically distributed over the surface of the protein, this leads to a dipole moment of about 315 debye (40). In all cases, the center of the interaction domain is located near the upper left of the exposed heme edge, close to the point at which the positive end of the dipole axis crosses the surface of the molecule (40,41). The present paper gives a precise definition of the interaction domain on cytochrome c for beef sulfite oxidase by comparison of the activities of singly modified CDNPI-lysine cytochromes c, and further characterizes the steady state kinetics of the system. Our results indicated that the site of the interaction for sulfite oxidase is nearly identical with those previously determined for the other enzymic electron exchange partners of cytochrome c. Preliminary accounts of this work were presented in 1979 (41,42). However, the assay conditions employed at that time did not allow differences to be observed between the various modified cytochromes c, large enough to distinguish between residues located in the interaction domain and those peripheral to it. A recent report (43), in which the same ionic conditions were employed to map this domain with trifluoroacetyl-and (trifluoromethy1)phenylcarbamyl-modified cytochromes c, concludes that lysines 8 and 25 directly participate in this interaction, whereas our findings place them peripheral to the enzymic domain.
Preparation of Beef Sulfite Oxidase-Beef sulfite oxidase was prepared from liver tissue according to the procedure of Johnson and Rajagopalan (22). The enzyme was prepared for the assay by dilution with 50% (v/v) ethylene glycol in 100 mM acetate (Tris), pH 7.5, containing 1% bovine serum albumin, divided into small aliquots, and stored in liquid nitrogen. Because thawing and freezing caused a decrease in activity, each aliquot of frozen dilute enzyme was used once. The concentration of sulfite oxidase was determined spectrophotometrically, using an extinction coefficient at 555 nm of 32 2nM-I cm" (23).
Beef Sulfite Oxidase Actiuity-The rate of reduction of cytochrome c was measured spectrophotometrically with a Beckman UV 5260 spectrophotometer at 416 nm (ernM = 40.3 rn"' cm"). Sodium sulfite and cytochrome c were added to the buffer to final concentrations of 2.5 mM and 0.3-3 I(M, respectively. The nonenzymic rate of reduction was monitored for 1.5 to 3 min. The enzyme on a plastic mixing plunger (Savant) was rapidly mixed into the cuvette (0.20 n~ final concentration). The reaction was followed to completion and the ' The abbreviation used is: CDNP-, 4-carboxy-2,6-dinitrophenylsubstituted.
linear initial portion of the time course afforded an accurate measurement of the initial steady state reduction of cytochrome c.
Binding of Ferricytochrome c to Sulfite Oxidase-Determination of the binding affinity of horse ferricytochrome c to purified beef sulfite oxidase was accomplished by the previously described (47) modification of the Hummel and Dryer (48) gel filtration technique. A column (0.7 X 50 cm) of Bio-Gel P30,200-400 mesh (Bio-Rad), was equilibrated in buffer containing various concentrations of cytochrome c, and a mixture of sulfite oxidase (2 nM) and excess cytochrome c (20 n~) in a volume of about 100 pl was applied to the column and allowed to filter through the gel. The amount of cytochrome c bound to sulfite oxidase was determined from the dithionitereduced spectrum according to the following relations: taking the extinction coefficients for sulfite oxidase at 555 nm and 550 nm to be 32 and 17 mM" cm", respectively, and the extinction coefficients for cytochrome c at 550 nm and 555 nm to be 29 and 10.5 mM-' cm", respectively. Calculation of the Dipole Moments of Native and Chemically Modified Cytochromes c-For the calculation of the dipole moments of various cytochromes c, it is assumed that at pH 7 lysine and arginine residues are positively charged. Aspartic acid, glutamic acid, the COOH-terminal carboxyl group, and the carboxyl group at the 4position of the CDNP moiety are considered to be negatively charged. The charge of the heme group is placed on the iron atom. In addition, charges of +05e and -0.5e are placed at the NH2-and COOHterminals, respectively, of four a-helices which are equal to or longer than six residues in length (49). Dipole moments are calculated using in which is the dipole moment, p and n are the number of positive and negative charges, respectively, F P and SN are radius vectors from the center of mass to the center of positive and negative charge, respectively, and e is the elementary charge.

Activity and Binding of Native Horse Cytochrome c-
Enzymic reduction of horse cytochrome c by beef cytochrome sulfite oxidase was studied as a function of pH in 50 m~ HC1 titrated with Tris, over the pH range 6.0 to 8.5. The results are depicted on an Eadie-Hofstee plot (Fig. 1). Raising the FH from 6.0 to 8.0 causes an increase in the maximal velocity, while the apparent Michaelis constant (K,) remains relatively unchanged. The increase in V,,, may reflect an increase in the rate of reduction of the bs heme, a process which is likely to be rate-limiting at high cytochrome c concentrations (see "Discussion"). This could arise from a change in either the rate of reduction of sulfite oxidase by sulfite which is protonated below pH 7.2, or an internal electron transfer step within the enzyme. However, such changes in the rate-limiting step would be expected to alter the apparent K , for cytochrome c. Since the K,,, does not vary, it would appear that other parameters may be involved.
The effect of increasing ionic strength on sulfite oxidase activity was determined using chloride (Tris), pH 8.0 buffer. As shown in Fig. 2 oxidase, direct binding measurements were conducted. The results (Fig. 3) are analyzed according to Hughes and Klotz (51). The negative reciprocal of the slope of the curve is the site dissociation constant (KD) for the binding of horse ferricytochrome c to beef sulfite oxidase. The dissociation constants varied from 1 X M at 10 mM to 1.5 X M at 50 m~, and the lines extrapolate to an r value of 2.0, indicating that two molecules of horse cytochrome c bind per bs heme of sulfite oxidase. Furthermore, it appears that the two sites are equal and noninteracting. However, the kinetics of reaction is monophasic, and whether one or two cytochrome c molecules are involved in the electron transfer process cannot be determined. While the binding of cytochrome c to sulfite oxidase is sensitive to increasing ionic strength, the KO values are approximately an order of magnitude larger than the apparent Michaelis constants for the reduction of cytochrome c by sulfite oxidase under the same ionic conditions. Activity of CDNP-Cytochromes c-The activities of 11 singly modified CDNP-cytochromes c were determined with beef sulfite oxidase. A stereodiagram of cytochrome c as viewed from the front, the surface containing the exposed heme edge, is shown in Fig. 4. The lysyl residues that have been derivatized and the heme are highlighted in black. Modification of the lysines on the front surface of cytochrome c-at positions 13,27,72,86, and 87-have previously been shown to interfere with the interaction of cytochrome c with beef cytochrome c oxidase and reductase, as well as yeast cytochrome c peroxidase (31-34, 40, 41). As shown in Fig. 5, the reaction of the CDNP-horse cytochromes c with sulfite oxidase displayed a wide array of activities. As is the case with the other enzymic interaction domains which have been mapped on cytochrome c, modification of lysyl residues on the front surface of the protein results in the greatest inhibition of the reaction. Cytochromes c modified at lysines slightly away from the front face-residues 7, 8, 25, and 73-exhibited intermediate activities, and derivatization of lysines on the back surface of the molecule, those a t positions 39 and 60, had the least inhibitory effect.
The kinetics of reaction of the CDNP-modified cytochromes c reported in Fig. 5

KD KD
where r is the ratio of cytochrome c bound to sulfite oxidase, [A] is the concentration of free cytochrome c in the equilibration buffer, KO is the dissociation constant, and n is the number of binding sites (51). Binding measurements were performed in HCI (Tris), pH 8.0 buffer, at the indicated ionic strengths. posed to lower ionic strengths at which the rate saturates at low cytochrome c concentrations, and higher ionic strength at which differences between the derivatives become minimal. An example of the kinetics obtained at 100 mM of Tris-chloride buffer is shown in Fig. 6, in which the activity of native horse cytochrome c at an ionic strength of 50 mM is shown for comparison. The difficulty of accurately measuring small differences in activity leads to an inability to readily distinguish between those lysyl residues that directly participate in the interaction of cytochrome c with sulfite oxidase from those that do not.

+-
Influence of the Dipole of Cytochrome e-Any disturbance of the charge configuration of cytochrome c will change the dipole moment of the molecule. If the electrostatic attraction between the enzyme and native cytochrome c is an important factor in aligning the protein as a productive enzyme-substrate complex, an alteration of the dipole moment will lead to a misoriented complex. For electron transfer to occur, the complex will have to be realigned, resulting in a higher than normal energy of activation. This extra activation energy can be calculated by assuming that it represents the work needed to rotate the cytochrome c in the electric field generated by the charges on sulfite oxidase through an angle 0 equal to that between the dipole moments of the native and modified cytochromes c (40). If the relative activities of CDNP-cytochromes c reflect only the change in dipole moment, they should fall on a line relating the logarithm of the relative activities to the amount of work needed to turn the dipole to form the productive complex, according to the following equation: in which r represents the relative activity; kmod/kN, the ratio of the intercepts with the Y-axis of a modified cytochrome c with respect to the native protein determined from the Eadie-Hofstee plot (Fig. 5); A , the pre-exponential Arrhenius factor; E,, the activation energy for the reaction of native cytochrome c with sulfite oxidase; U, the extra activation energy required for turning the dipole of a modified cytochrome c such that a productive complex is formed; k , Boltzmann's constant; T, the temperature; E , the electric field.' The results of such an analysis are shown in Fig. 7. This demonstrates that the proteins modified at lysines 60, 39, 7, 25, 8, and possibly 73 and 87, have activities with sulfite oxidase that can be accounted for entirely by the change in dipole moment. This is caused by the derivatization, which neutralizes the positive charge of the €-amino group and introduces a negative charge at the 4-position of the CDNP aromatic ring. In contrast, the activities of the cytochromes c modified at residues 13, 27, 72, and 89 are clearly lower than would be expected from the alteration in their dipole moments.

DISCUSSION
From the steady state kinetics and binding results, it can be seen that the apparent Michaelis constant, determined at saturating sulfite concentration, is much smaller than the dissociation constant determined by gel filtration. This discrepancy may be explained by considering the following scheme: If k4, the rate of generation of the form of the enzyme that is capable of reducing cytochrome c, is rate-limiting at high cytochrome c concentrations, then it follows that as the cytochrome c concentration is increased, an increasing fraction of the enzyme is converted to the state (E') which is unable to reduce cytochrome c. E' is likely to represent the enzyme with the b5 heme in the ferric form, although other forms incapable of reducing cytochrome c could occur. This leads to saturation of the kinetics of reaction at concentrations of cytochrome c well below the concentration at which the binding of cytochrome c to sulfite oxidase is saturated. Further support for this explanation of the kinetics comes from the maximal turnover number observed for sulfite oxidase activity of approximately 50 electrons per s. This is a rather slow turnover as compared to the reaction of cytochrome c with purified mitochondrial cytochrome c reductase, which under similar conditions yields maximal turnover numbers ranging from about 500 to 800 electrons/s (33), indicating that cytochrome c is capable of much more rapid turnover than is observed with sulfite oxidase. The small turnover number for this reaction is unlikely to be due to a slow rate of dissociation of ferricytochrome c from the enzyme, since the enzyme binds only weakly to cytochrome c. This is in contrast to what has been observed with cytochrome oxidase to which cytochrome c binds very tightly, leading to a rate-limiting dissociation under appropriate ionic strength conditions (52,53). This would also explain the observation that increasing ionic strength with chloride (Tris) buffer gives strictly competitive inhibition of the reaction of cytochrome c with sulfite oxidase, even though the reaction of sulfite with sulfite oxidase under the same conditions shows mixed noncompetitive inhibition (27).
However, the CDNP-cytochromes c display a wide array of activities indicating that the apparent K, is strongly affected by the binding of cytochrome c to sulfite oxidase. Although V,,, may be determined by a rate-limiting step other than the reaction with cytochrome c, saturation of the reaction rate will depend on the affinity of the cytochrome c for the enzyme, and the CDNP-modified cytochromes c apparently have different affinities. To distinguish between lysyl residues which are located in the interaction domain, and those removed from the domain, it was proposed that the inhibition caused by modifying a given lysine results from an alteration of the dipole moment of the molecule and of any direct interaction between cytochrome c and its oxidation-reduction partner at that residue (40). From the activities of the CDNP-cytochromes c with beef sulfite oxidase, and the previous localization of interaction domains on cytochrome c for beef cytochrome c oxidase and reductase, as well as yeast cytochrome c peroxidase, it was concluded that electron transfer occurs via the exposed heme edge on the front surface of the molecule. This site represents only 0.6% of the total surface area of the protein accessible to water as calculated from the X-ray crystallographic structure of the protein (54). If the site of electron transfer on sulfite oxidase represents an equivalently small proportion of the total surface area of that molecule, then in random collision, the two molecules have a probability of less than 4 X of being in the correct orientation for electron transfer. Even if this estimate of the probability is in error by 3 orders of magnitude, the process would still be too inefficient to explain the very fast observed bimolecular rate constants determined for the reaction of cytochrome c with several of its mitochondrial oxidation-reduction partners (55)(56)(57)(58), and suggests that electrostatic forces are involved in increasing the efficiency of the reaction. Indeed, the bimolecular rate constant for the reaction of cytochrome c with another b5 heme-containing enzyme, yeast lactate-cytochrome c reductase, has been directly determined to be close to that expected for a diffusion-controlled reaction between two proteins (56), indicating that the reactants must orient as they approach each other.
Horse ferricytochrome c has a net charge of +9e and, if we assume that beef sulfite oxidase has a net negative charge (primary structure determinations on the b5 heme domain from rat and chicken sulfite oxidase (23, 59) have demonstrated that this fragment of the protein is indeed acidic), then these net charges wiU serve to bring the two molecules together. It should be noted, however, that although the energy of this interaction is of longer range than other electrostatic forces, being proportional to l / r ( r representing the distance between the centers of the two molecules), there are no directional forces involved. Therefore, this type of attraction is not sufficient to ensure correct alignment of the proteins.
To increase the probability of formation of a productive electron transfer complex, other electrostatic forces may be involved. It has been suggested that the asymmetric distribution of charges on the surface of cytochrome c is of physiological importance and aids the molecule in attaining the correct orientation for electron transfer (40,60). The energy of interaction between a net charge and a dipole shows a l/r2 dependence, indicating that cytochrome c will orient its dipole with respect to a net negative charge as the two molecules approach. Additionally, if beef sulfite oxidase has a dipole moment this would further serve to align the two proteins prior to formation of a complex. However, at this point there is no way of assessing whether such a dipole exists for sulfite oxidase since neither the complete amino acid sequence nor the tertiary structure of the protein is known.
The CDNP-cytochrome c derivatized at a lysyl residue directly involved in the interaction of cytochrome c with beef sulfite oxidase exhibit relative activities which cannot be totally accounted for by the alteration of the dipole moment of the protein (see Fig. 7). The points for the cytochromes c modified at lysines 13, 72, 86, and 27 are far below the line, indicating that these residues form part of the interaction domain. Residues 73 and 87 may also be involved, but not to a great extent. On the other hand, the activities of the cytochromes c modified at positions 60, 39, 7, 25, or 8 can be explained in terms of their altered dipole moments. Based on these results, a domain on cytochrome c for the interaction with beef sulfite oxidase was drawn (Fig. 8). This domain is very similar to those determined for the interaction of cytochrome c with beef cytochrome c oxidase and reductase, and yeast cytochrome c peroxidase (40,41).
From kinetic studies, in which trifluoroacetyl-substituted and (trifluoromethy1)phenylcarbamyl-substituted lysine cytochromes were employed (43), it was concluded that lysyl residues 8, 13, 25, 27, 72, 79, 86 (by implication), and 87 are within the interaction domain. The result is at variance with ours, which show that residues 8 and 25 do not appear to be directly involved with the interaction. There could be two reasons for this discrepancy. First, the negatively charged CDNP-lysine residue may fold away from the enzyme because of electrostatic repulsion, whereas neutral trifluoroacetylsubstituted or (trifluoromethyl)phenylcarbamyl-substituted groups at the same location would not be repelled, and only interfere by steric hindrance. Secondly, the interaction domain may have been overestimated, since at the ionic strength employed, the differences in activity are too small to be easily use the contribution of a charge on a given lysyl residue to the total electrostatic energy of interaction between two protein molecules, in order to calculate a distance between this lysine and its supposed countercharge on the surface of the other protein, neglecting all other charges. (c) For the calculation of the distances mentioned above, they use Debye's formula (61) outside its validity range. The quoted examples which purport to show that the Debye-Huckel treatment is valid at high ionic strength (0.5-1.5 M), are based not on the classical Debye-Huckel theory (62), but on extensions that involve addition of a term to the formula for the activity coefficient which is linear in ionic strength and contains an empirical parameter (63). This additional term is not included in their analysis.
As mentioned above, the primary structures of the cytochrome b5 heme fragment from rat and chicken sulfite oxidase have been determined (23,59). It has been proposed that this portion of the sulfite oxidase molecule, along with microsomal b5 and the cytochrome bp fragment of yeast lactate-cytochrome c reductase, are part of a cytochrome b5 superfamily (64) and share a common tertiary structure (the cytochrome b5 fold). These heme binding domains have extensive sequence homology (63), which, given their diverse origins, strongly suggests that they are indeed related evolutionarily. Salemme (65) has proposed a model for the interaction of cytochrome c with microsomal cytochrome b5 based on the crystallographic structures for both proteins. A comparison of the sequences of the cytochrome b5 domains of rat and chicken sulfite oxidase with that for microsomal cytochrome bs, shows that, although both rat and chicken sulfite oxidase are very The Reaction of Cytochrome c with Sulfite Oxidase acidic fragments, they lack several acidic residues present in microsomal b5, located near the exposed heme edge. Salemme has proposed that four acidic groups on microsomal b5 are involved in charge pairing with cytochrome c. Of these four charges, only two are present in rat and chicken sulfite oxidase, indicating that fewer charge interactions may be involved in the binding of cytochrome c to sulfite oxidase. Our direct binding measurements demonstrate that the binding of cytochrome c to beef sulfite oxidase is relatively weak, and we are currently investigating the interaction of cytochrome c with microsomal b5 to determine if indeed there are significant differences in activity and binding.