Background
What's New?
Recent data (Mason et. al, 2006) now proves that a purely competitive model for NO inhibition of CCO is insufficient for fitting known data over a wide range of oxygen tensions and enzyme turnover rates.
Calculation of CCO Ki (NO) at High Enzyme Turnover
The purpose of this experiment was to re-test the previously proposed theory that NO can outcompete oxygen by binding to both the fully reduced and singly reduced binuclear center of CCO.
NO inhibition of CCO was compared to its inhibition by carbon monoxide (CO). Like O2, CO can bind to reduced heme iron only when the adjacent copper is also reduced, meaning that CO inhibits CCO in a purely competitive manner. The apparent Km (O2) was measured at various concentrations of both inhibitors. The true Ki for each inhibitor calculated from this data was 0.2nM for NO and 0.3uM for CO. These results show that inhibition by NO at high enzyme turnover is 1,000 to 2,000 fold more effective than inhibition by CO and support the hypothesis that NO does not inhibit CCO in a purely competitive manner.
Reassessment of Hill Coefficients for NO Binding
The Hill coefficient is calculated by plotting the log of the concentration of NO versus the fractional inhibition of CCO. Data from Mason et. al (2006) show that the Hill coefficient is not a constant, but rather depends on the enzyme turnover. Figure 11 shows that when enzyme turnover is low, the Hill coefficient is greater than 1, but, as enzyme turnover increases, the Hill coefficient approaches 1.
Figure 11: Hill coefficients for NO inhibition as a function of enzyme turnover. (Mason et al, 2006)
Since the Hill coefficient for NO inhibition is not constant and is not 1, NO does not inhibit CCO in a purely competitive manner.
Mode of Inhibition by NO: Simple Competitive Inhibition or More Complex?
A detailed kinetic analysis of the oxygen competition of NO inhibition was performed to determine if NO binds CCO purely in competition with oxygen (Figure 12).
Figure 12: The apparent IC50 (NO) versus [O2] for high and low enzyme turnover and fit with lines corresponding to a simple competitive model of inhibition. (Mason et al, 2006)
The apparent IC50 (NO) was calculated as a function of the concentration of oxygen at both high and low enzyme turnover. The data for both cases is shown fitted with lines that correspond to a simple competitive model of inhibition (Figure 12). The y-intercept is the true Ki 0.2nM. The plot clearly shows that, at high enzyme turnover, the data is consistent with the pure competitive model. However, at low enzyme turnover, inhibition by NO, while still sensitive to oxygen concentration, is not purely competitive.
Figure 13: The apparent IC50 (NO) versus [O2] for high and low enzyme turnover and fit with lines corresponding to the expanded model of inhibition proposed by (Mason et al, 2006)
Figure 13 shows the data fitted using Mason et al.'s expanded model of inhibition. In this model NO can interact both competitively or noncompetitively with oxygen. NO reacts with the reduced heme iron of the binuclear center in a competitive manner with oxygen. The expanded model also includes noncompetitive inhibition which occurs when NO reacts with the oxidized copper of the binuclear center, resulting in oxidation of NO to nitrite.
Figure 13 clearly shows that by including the noncompetitive interaction of NO with the oxidized CuB, the data can be fitted at all rates of enzyme turnover.