States of amino acid residues in proteins: VI. Heme-linked histidine residues in cytochrome c as determined with diazonium-1-H-tetrazole

Abstract

Diazonium-1-H-tetrazole(DHT), a new coupling reagent for histidine residues, was reacted with horse heart and baker's yeast cytochrome c to differentiate between free and heme-linked histidine residues. Coupling to the histidine residues in horse heart cytochrome c proceeds in three steps with increasing DHT concentration, such that each of the three histidine residues in the molecule is coupled at a different DHT concentration. Coupling to the four histidine residues in the yeast cytochrome c molecule also proceeds in three steps. Two of the residues are coupled in the first step, and each of the remaining two residues at higher DHT concentrations in the second and in the third steps, respectively. The Soret band of these cytochromes does not change in the first step when the most reactive histidine residue(s) is coupled, but changes stepwise in parallel with the second and the third stages of coupling. Reduction of ferricytochrome c appreciably decreases the reactivities toward DHT of the two residues responsible for the second and the third steps, but affects slightly or not at all the reactivity of the most reactive residue(s). Dihistidyl coordination to the iron atom of heme is proposed from these results. The two iron-linked histidine residues were located at positions 18 and 26 in the amino acid sequence by digestions with trypsin and with Nagarse of DHT-treated horse heart cytochrome c, in which only the most reactive histidine residue was modified with DHT.