A re-evaluation of some basic structural and functional properties of Pseudomonas cytochrome oxidase

Abstract

Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of epsilon(ox.) (410)=282x10(3) litre.mol(-1).cm(-1) was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d(1) components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d(1) bound NO. Titration experiments on the reduced protein over the pH range 5-7, with nitrite as a precursor of NO, showed that the haem d(1) had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.

Activation of the Cytochrome cd1 Nitrite Reductase from Paracoccus pantotrophus

“…The addition of NO to oxidized cd 1 from both Pa and Ps also results in absorption spectra similar to that of Ps cd 1 *-X (Fig. 1b) (19,49). This would, at first, appear to contradict our data because ferric nitrosyl hemes are best described as diamagnetic Fe II -NO ϩ species (50) This interpretation would require that a transient protein radical is formed during the reaction but dissipates rapidly.…”

“…It therefore appears that all Fe III heme d 1 is in the low-spin state following reaction with NO 2 Ϫ . The absorption spectrum of Pp cd 1 *-X is very similar to those of the oxidized cytochromes cd 1 from Pa and Ps, enzymes in which the heme d 1 is fully low-spin at all temperatures (5,19). This reactivity of oxidized Pp cd 1 is unexpected given that it is generally accepted that this state does not interact with NO 2 Ϫ (34).…”

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

“…11. NIR activity was measured anaerobically at 27°C in 50 mM sodium phosphate buffer (pH 6.2), either after oxidation of reduced azurin (12) or during amperometric measurement of NO production with a NO electrode (ISO-NO; World Precision Instruments, Sarasota, FL). A typical experiment at the electrode was carried out at 25°C in the presence of ascorbate (13 mM) and N,N,N,N-tetramethyl-p-phenylenediamine (0.1 mM) as electron donors and initiated by adding different concentrations of nitrite (10-1,200 M).…”

NO Production by Pseudomonas aeruginosa cd1 Nitrite Reductase

“…the nitrite anion bound to the d 1 heme iron, followed by protonation and dehydration. Product inhibition may occur, since the NO produced at the catalytic site forms stable complexes with the ferrous d 1 heme and, at acidic pH, also with the c heme (26). A possible reaction scheme for cd 1 NIR, shown in Figure 2, assumes that functional interactions between the two monomers can be neglected, which may not necessarily be the case.…”

“…It is worthwhile mentioning that the steady state oxidation of macromolecular substrates by Pa-cd 1 NIR with nitrite as electron acceptor is much slower at pH above 6.5 (26). Thus, under pre-steady state conditions at pH 6.2, the enzyme is locked in the NO inhibited form after one catalytic cycle in the presence of excess reducing equivalents (29).…”