Functional intermediates in reaction of cytochrome oxidase with oxygen.

Abstract

The development of a low temperature kinetic method for the flash photolysis of the compounds of membrane-bound cytochrome as with carbon monoxide in the presence of oxygen affords evidence for three categories of functional intermediate compounds of cytochrome a3 and oxygen. The three classes are identified as follows: Compounds of Type A are considered to be "oxy" compounds of the ferrous heme. They have the composition asQ-0,. Compounds of Type B are considered to be peroxide compounds (Cu2+ass+-02. or Cul+a:,+. 02H2) or the equivalent heme Fe-Cu peroxide bridge structures. Compounds of Type C are formed from the ferricyanide pretreated oxidase and may involve higher oxidation states of the heme iron such as quadrivalent iron, and peroxide. Kinetic and equilibrium studies show these compounds to be functional in oxygen reduction in the sequence A B -cytochromes a, c, cl, etc.The observation of intermediates in enzyme-substrate interactions has led to a verification of the Michaelis-Menten theory upon which enzyme mechanisms are based (1-5). The most likely enzyme-substrate compounds (6) to be discovered first are those that are abortive, nonfunctional, or "dead-end." History tells us that Kurt Stern's (7) red enzyme-substrate compound in the catalase-ethyl hydrogen peroxide reaction was discovered only because it was inactive; the active compound was too labile (2) for the techniques at hand. Peroxidase was also found to form an inactive stable enzyme-substrate compound IV (8) which has recently been restudied by Yamazaki (9).A similar development seems to have occurred in the case of cytochrome oxidase, where Okunuki (10), Lemberg (11), Wainio (12), and others have studied what they term an "oxygenated" form of the oxidase. The possibility that the "oxygenated" form could be preceded by more active forms, just as in the case of the catalases and peroxidases, has led us to the current study.Gibson and Greenwood have studied the detergentsolubilized oxidase and have discovered a time separation of components a a, and Cu (13). However, heterogeneity is present in such preparations and thus the membrane-bound oxidase of intact mitochondria has been used in these studies.Some success with this approach was obtained in experi- Of greatest advantage in these studies was the fact that the frozen mixture of oxygen and the carbon monoxide compound of cytochrome oxidase can be chilled prior to photolysis to the point where the half-time of the reaction following photoly-

The involvement of the fully oxidized state in cytochrome oxidase reaction with oxygen studied with the 655 nm band as a probe

“…Discussion tic of the a3 form [10] we have demonstrated on the hand that the reaction with oxygen of mitochondrial cytochrome oxidase in the mixed valence state does not lead to the formation of the fully oxidized state. This result is consistent with the prominent absorbance increase observed at 606 nm [4,5,12] when Compound C forms which strongly supports a reduced state of the iron. These considerations have suggested 2 + the formulation of Compound C as a3 -0g -Cu 2+ 3+ [9].…”

“…On the other hand we have shown that a3 forms before the complete disappearance of Compound A expressed by the absorbance decrease at 590 nm on fig.2. The results in fig.2 provide evidence that cytochrome c oxidation may occur at temperatures lower than -50°C previously suggested as a limit [5]. The oxidation of cytochrome c implies the one of cytochrome a to a certain extent.…”

“…One questionable point has been the redox level of hemes a and a 3 in the stage of Compound C and Compound B formation. Several identifications have been proposed [4][5][6][7][8][9] which did or did not involve the fully oxidized state. On the basis of their heme-heme interaction model, Wikstrom et al [8] suggested that the mechanism of oxygen reduction by cytochrome oxidase does not involve the fully oxidized form.…”

“…The use of a low temperature technique as developed by Chance et al [1] led to the discovery of intermediates in the reaction of cytochrome oxidase with oxygen [2][3][4][5]. The reaction is initiated by photolysis of CO bound mitochondria in presence of oxygen.…”

X‐ray absorption edge studies on cyanide‐bound cytochrome c oxidase

“…Recently, there has been a great deal of interest in the chemical and physical properties of various inhibitor complexes of cytochrome c oxidase [3][4][5][6][7]. In particular, considerable effort has been spent on studying the cyanideand carbon monoxide-bound protein with the hope of trapping one or more of the metal components in the oxidized or reduced form [8][9][10][11]. The rationale for such studies is the possibility that 'mixed valence' forms of the CN-or CO-inhibited protein might provide some clues about the mechanistic pathway of electron transfer to oxygen as well as the interaction of the four metal components in the functional protein.…”