The reactions of Pseudomonas cytochrome c-551 oxidase with potassium cyanide

Abstract

The binding of cyanide to both oxidized and ascorbate-reduced forms of Pseudomonas cytochrome c-551 oxidase was investigated. Spectral studies on the oxidized enzyme and its apoprotein showed that the ligand can bind to both the c and d, haem components of the molecule, and kinetic observations indicated that both chromophores reacted, under a variety of conditions, with very similar rates. Cyanide combination velocities were dependent on ligand concentration, and increasing the pH also accelerated the reaction; the second-order rate constant was estimated as approx. 0.2M-1 . s-1 at pH 7.0. The binding of cyanide to the protein was observed to have a considerable influence on reduction of the enzyme by ascorbate. Spectral and kinetic observations have revealed that the species haem d13+-cyanide and any unbound haem c may react relatively rapidly with the reductant, but the behaviour of cyanide-bound haem c indicates that it may not be reduced without prior dissociation of the ligand, which occurs relatively slowly. The reaction of reduced Pseudomonas cytochrome oxidase with cyanide is radically different from that of the oxidized protein. In this case the ligand only binds to the haem d1 component and reacts much more rapidly. Stopped-flow kinetic measurements showed the binding to be biphasic in form. Both the rates of these processes were dependent on cyanide concentration, with the fast phase having a second-order rate constant of 9.3 X 10(5) M-1 . s-1 and the slow phase one of 2.3 X 10(5) M-1 . s-1. The relative proportions of the two phases also showed a dependency on cyanide concentration, the slower phase increasing as the cyanide concentration decreased. Computer simulations indicate that a reaction scheme originally proposed for the reaction of the enzyme with CO is capable of providing a reasonable explanation of the experimental results. Static-titration data of the reduced enzyme with with cyanide indicated that the binding was non-stoicheiometric, the ligand-binding curve being sigmoidal in shape. A Hill plot of the results yielded a Hill coefficient of 2.6.

The nitrite reductase from Pseudomonas aeruginosa : Essential role of two active-site histidines in the catalytic and structural properties

“…The 3D structure of the cyanide derivative of reduced P. pantotrophus NIR (23) shows that the two His and the Tyr in the active site are within H-bonding distance of cyanide, suggesting that a positive electrostatic potential in the pocket is essential for stabilization of the bound anion. An unusually high positive charge density has also been found in the heme pocket of the Escherichia coli sulfite reductase, an enzyme capable of binding both sulfite and nitrite with high affinity, catalyzing their reduction to sulfide and ammonia, respectively (26). The unique role of His-369 in stabilizing the enzyme-substrate complex, although unexpected, seems to be in agreement with the structural data shown in Fig.…”

Bacterial nitrite‐reducing enzymes

“…The high redox potential is consistent with the ability of ascorbate to reduce this enzyme. When P. ueruginosu cytochrome cd, is in the ferric state, both the c and dl haern can bind cyanide (Barber et al, 1978). Cyanide binding to haem c occurs by displacement of the methionine axial ligand and an interaction between the c haem and the dl hacm is indicated by a small change in the EPR spectrum of the c haem on the binding of cyanide to haem dl (Suthcrland et al, 1986).…”

“…MCD studies of the cyanide derivative showed d, to be low-spin (Walsh et al, 1979). Carbon monoxide and cyanide inhibit the oxygen reductase activity of this enzyme, but only cyanide appears to inhibit the nitrite reductase activity (Barber et al, 1978;Parr et al, 1974). Thc reaction of cytochrome cd, nitrite reductase with CO has bccn studied by static titration, stopped-flow and flash photolysis ( P a r et al, 1975).…”

“…Cyanide binding to haem c occurs by displacement of the methionine axial ligand and an interaction between the c haem and the dl hacm is indicated by a small change in the EPR spectrum of the c haem on the binding of cyanide to haem dl (Suthcrland et al, 1986). When cytochroine cd, is reduced, haem d, can bind CO (Parr et al, 1975), cyanide (Barber et al, 1978) and metabisulphite (Parr et al, 1974). MCD studies of the cyanide derivative showed d, to be low-spin (Walsh et al, 1979).…”

Dissimilatory Nitrite and Nitric Oxide Reductases

“…[41][42][43][44][45][46][47][48][49] Many date from the days when the enzyme was referred to as "bacterial cytochrome oxidase", because of its ability to reduce O 2 to water (albeit much more slowly than mammalian cytochrome c oxidase) and the presence of four electron acceptor groups per dimer. These studies were aimed at exploring the relationship between the bacterial and mammalian cytochrome oxidases.…”