Table 1.

Kinetic properties of the WT and mutant enzyme

Unit	kcatapp under 1 atm of H2*, s−1	Km†, m atm (H2)	kcat‡, s−1	kinCO§, s−1 atm(CO)−1	koutCO§, s−1	kout/k¶
WT	750 ± 90	10	760 ± 95	1 × 104 to 2 × 104	2 to 10	1.25
FI	800 ± 30	50	840 ± 50	2 × 103	4 × 10−1	10−1
MM	590 ± 30	200	675 ± 90	65	4 × 10−3	2 × 10−2

*Apparent first-order rates constants for H₂ oxidation, determined in solution assays in the presence of 1 atm H₂ and 50 mM oxidized methyl viologen, at T = 30°C, pH = 8. A turnover frequency of 1 s⁻¹ is equivalent to 0.66 μmol of H₂ oxidized per min and per mg of enzyme.

^†Michaelis constant relative to H₂ (21). The accuracy is of the order of 50%.

^‡Values of k_cat extrapolated using k_cat = k_cat^app (1+K_m/[H₂]).

^§Rate constants relative to CO binding (k_in^CO) and release (k_out^CO) determined from fitting data such as those in Fig. 3 (27). For the WT, the values have been extrapolated to 30°C from the data in Fig. 4. ″One atm of CO″ refers to the concentration of CO dissolved in a solution that is equilibrated under 1 atm of CO at 25°C.

^¶Rate of dihydrogen release from the active site divided by the rate of H⁺/D⁺ exchange at the active site, determined from interpreting isotope exchange assays such as those in Fig. 5. We used the model and the method that corrects for the slow gas consumption by the mass spectrometer, both described in SI Text.