Distinction between oxidizing and reducing sites of cytochrome C by chemical modification with pyridoxal phosphate

It was recently reported that antibodies to cytochrome c inhibit selectively the oxidation of the enzyme by cytochrome oxidase and its reduction by succinic dehydrogenase [ I] . However, attempts to differentiate between oxidizing and reducing sites of cytochrome c through chemical modification have been hitherto inconclusive. In this communication, we describe the preparation of cytochrome c derivatives obtained by covalent binding of pyridoxal phosphate, with normal physicochemical properties and reactivity with cytochrome oxidase, but with impaired enzymic reducibility.


Introduction
It was recently reported that antibodies to cytochrome c inhibit selectively the oxidation of the enzyme by cytochrome oxidase and its reduction by succinic dehydrogenase [ I] . However, attempts to differentiate between oxidizing and reducing sites of cytochrome c through chemical modification have been hitherto inconclusive.
In this communication, we describe the preparation of cytochrome c derivatives obtained by covalent binding of pyridoxal phosphate, with normal physicochemical properties and reactivity with cytochrome oxidase, but with impaired enzymic reducibility.

Materials
Horse heart cytochrome c (Type III), NADH, NADH-cytochrome c reductase, and pyridoxal phosphate (PLP) were from Sigma Chemical Co. Beefheart cytochrome oxidase was prepared according to Yonetani [2]. All other chemicals were commercial preparations of the best degree of purity available.

Methods
The enzymic activity of native and modified cytochrome c was estimated by measuring their ability to restore the respiratory activity to cytochrome c depleted rat liver mitochondria [3]. Spectrophotometric measurements of ferrocytochrome c oxidation by cytochrome oxidase were performed as described by Smith [4]. Initial rates of reduction of ferricytochrome c by rat liver mitochondrial succinic dehydrogenase were followed at 550 nm, in the presence of 1 mM KCN [S] . NADH-cytochrome c reductase activity was assayed according to Mahler [6]. Covalent binding of PLP to cytochrome c was performed in the following way: a solution of ferric cytochrome c (5 mg/ml) in 0.05 M Tris-cacodylate buffer, pH 7.0 was incubated for 5 min in the presence of 1-l .5 mM PLP. A solution of 0.4 M NaBH4 in 0.02 M NaOH was added, to make a final concentration of 25 mM borohydride. After 10 min at room temperature, the solution was diluted lo-fold and oxidized with potassium ferricyanide. Chromatography on Amberlite CG-50 using a NaCl gradient as eluent resolved the products into 3 peaks ( fig. 1). The slowest peak was native cytochrome c. The first and second fractions showed an absorption band at 325 nm. The absorbance at 325 nm indicated that the first fraction contained two residues of PLP, and the second fraction only one. They were labelled (PLP)2-c and (PLP)I-c respectively.

Results
The two derivatives showed the characteristic properties of native cytochrome c. In the ferric form, the 695 nm band was present, indicating coordination of the iron by a methionine sulfur. The rate constants for the reduction with ascorbate at pH 7. (PLP)2-c were not autoxidizable, the ferric forms reacted with cyanide at pH 7, with rates identical to those of native cytochrome c. All these results indicated that in (PLP)l-c and (PLP)2-c the heme environment remained the same as in native cytochrome c, and that the chemical modifications did not cause major alterations in the tertiary structure of the protein. The restoration of respiratory activity of cytochrome c depleted mitochondria by (PLP)l-c, (PI_Q2-c and the native protein is shown in fig. 2. (PLP)l -c is only partly active; (PLP),-c was almost entirely inactive. On the other hand, the two derivatives were oxidized by a purified preparation of beef heart cytochrome oxidase at identical rates (table 1). This indicated that the reducibility of (PLP)r -c and (PLP)2-c by enzyme systems was impaired and, indeed, this was found to be so for succinic dehydrogenase of the respiratory chain and for NADH cytochrome c reductase (table 1).
In order to determine the sites of covalent binding of PLP to the cytochrome c protein, (PLP)l-c and (PLP)2-c were digested with chymotrypsin, and chro- matographed on Dowex 50-X4 at 55°C with pyridineacetate buffers. The fractions showing absorption peaks at 325 nm were further purified by paper chromatography and analyzed for amino acid content. The compositions of the PLP-bound peptides permit to identify them with the following partial sequences: l-10; 68-74, and 83-94. The main heme peptide isolated showed an increase in absorbance in the 325 nm region, although not a definite peak. The possibility that PLP is bound to it is being investigated. For this reason, the relative yields of the different PLPpeptides in the two modified proteins could not be estimated.

Discussion
The covalent binding of one or two PLP molecules affects the enzymic reducibility of cytochrome c, while its enzymic oxidizability and its reduction with ascorbate are not impaired. This is, to our knowledge, the first instance of a modification of the cytochrome c protein that distinguishes between reductase and oxidase sites of the enzyme, without, at the same time, causing major changes in the physicochemical properties of the molecule.
In cytochrome c, the amino terminal end is acetylated, hence only lysine residues are susceptible to FEBSLETTERS October 1973 Table I  Enzymic properties  The PLP-bound peptide 68-74 contains lysines 72 and 73, that belong to the invariant 70-80 sequence, and are located at a different position in the molecular surface [7].
The following conclusions can be reached: first, that the chemical modification with PLP has singled out two regions of cytochrome c as possible sites for its attachment to the mitochondrial reductases; second, that these regions cannot be involved in the reaction with cytochrome oxidase. Studies now in progress will hopefully define the exact location of the reductase site of cytochrome c.