Stopped-flow analyses of the reactions of oxy-DevS with substoichiometric NO show conversion of the oxy complexes to Fe(III) hemes (). Specifically, while the initial oxy-DevS solution exhibits characteristic absorptions at 414, 541, and 577 nm (see Materials and Methods), the first stopped-flow trace obtained 4 ms after mixing of oxy-DevS with NO, and subsequent traces, show a decrease of these absorbance features in favor of a Soret maximum at 407 nm and absorption at 498 and 632 nm that are consistent with formation of Fe(III)-DevS. Despite our efforts to maximize the anaerobicity of the stopped-flow instrument, reproducing precise NO concentrations in the low micromolar range was unworkable in the absence of an anaerobic chamber confining the apparatus. Consequently, NO concentrations were inferred from the amount of oxy-complex consumed after mixing. At 2 μM NO, the rate of the reaction with oxy-DevS at pH 7.5 was observed to be biphasic with kobs
= 36 s-1
and 1.2 s-1
(). Analysis of total nitrate and nitrite concentrations confirm quantitative conversion of NO to NO3-
by oxy-DevS (data not shown), as observed previously for oxymyoglobin and M. bovis
). Thus, in spite of technical limitations preventing adequate measurements to determine second order rate constants of this reaction, the existing results clearly demonstrate efficient NOD reactivity in the sensor kinase DevS.
Stopped-flow UV-vis absorption spectra of the reaction of 4 μM oxy-DevS with 2 μM NO at 4.2 °C and pH 7.5. Inset: kinetics of the reaction measured at 407 nm.
The NOD reaction in DevS was also monitored by RFQ and RR spectroscopy, techniques that we have used together successfully to trap and characterize an Fe(III)-nitrato complex in the NOD reaction of myoglobin at pH 9.5 (23
). The high-frequency RR spectra of 300 μM oxy-DevS mixed with excess NO and frozen at 7, 15, and 55 ms are nearly identical to that of the oxy-complex (). Specifically, the porphyrin ν4
, and ν10
modes are observed at 1374, 1501, 1581 and 1640 cm-1
, respectively, and are characteristic of six-coordinate low-spin (6cLS) Fe(III) heme species (24
). In contrast, the Fe(III) heme of DevS is predominantly 6cHS with ν4
, and ν10
at 1370, 1478, and 1617 cm-1
Figure 2 High-frequency RR spectra of RFQ samples of the reaction of 300 μM oxy-DevS with excess NO compared to those of resting oxy- (top trace) and Fe(III)-DevS (bottom trace). (λexc = 413 nm, 20 mW; sample temperature ~ 105 K). The 1473 (more ...)
The low-frequency RR spectra of the RFQ samples show a rapid consumption of the oxy complex, as determined by the loss of the ν(Fe-O2
) at 566 cm-1
in the 7-ms RFQ sample (); This mode was previously observed at 563 cm-1
in wt DevS at room temperature (10
). The loss of the ν(Fe-O2
) band is accompanied by the gradual appearance of a mode at 601 cm-1
, which downshifts by − 5 cm-1
when the NO solution is substituted with 15
NO (). The observed frequency and 15
N-isotope shift are consistent with the assignment of this mode to a ν(Fe-NO) from an Fe(III)-NO complex (26
). These assignments are consistent with the RR spectra of resting oxy-DevS, Fe(III)-DevS, and Fe(III)-NO DevS (). Thus, the RR spectra of the RFQ samples provide evidence for the rapid decay of the oxy complex upon its reaction with excess NO to generate an Fe(III) state that can further react with excess NO to form a stable Fe(III)-NO complex.
Figure 3 Low-frequency RR spectra of RFQ samples of the reaction of 300 μM oxy-DevS with excess NO compared to those of resting oxy- (top trace), and Fe(III)NO-DevS and Fe(III)-DevS (bottom two traces). The spectrum of an RFQ sample frozen after 55 ms (more ...)
The RFQ-RR data suggest that Fe(III) DevS has an unusually high affinity for NO, and indeed, titration of an anoxic solution of Fe(III) DevS with a solution of NO yields an apparent Kd
of ~ 5 μM (). This unusually high NO affinity exhibited by an Fe(III) heme protein (28
) suggests that the Fe(III)-NO state could be physiologically relevant in the infected macrophage. The Fe(III)-NO complex of DevS shows only marginal activity in autophosphorylation assays compared to oxy-DevS () (8
), but we have found that the Fe(III)-NO complex is easily reduced with 10 mM ascorbate, i.e., about 20-fold faster than the Fe(III) DevS state (). Thus, reduction of the Fe(III)-NO complex to Fe(II)-NO can be expected to be a rapid process in the reducing cellular environment and would lead to the activation of the kinase domain. As such, the NOD reaction of oxy-DevS may represent a rapid switching mechanism to convert an inactive oxy state to an active Fe(II)-NO species via formation of the Fe(III) and Fe(III)-NO DevS states ().
Binding curve determined from the titration of anoxic Fe(III)-DevS with NO (A), and reduction time course of 4.5 μM Fe(III)-DevS with 10 mM ascorbate in the presence and absence of 25 μM NO (B).
Autophosphorylation of 5 μM DevS in the presence of 500 μM ATP.
Figure 6 Proposed mechanism of kinase activation in the reaction of oxy-DevS with NO. The kinase domain is color grey when inhibited, orange for the intermediate autokinase activity observed in deoxy-DevS, and red for the full activation observed with the Fe(II)-NO (more ...)
Given the possible early sensing role of DosT, the reactivity of its oxy form with NO and the NO affinity of its Fe(III) state are also of interest. Preliminary stopped-flow monitoring of the reaction of oxy-DosT with NO (at 0.57 μM) revealed a monophasic reaction with kobs ~ 1 s-1 similar to the slower component of the biphasic rate observed in DevS (data not shown), but no further stopped-flow experiments were carried out with DosT because activity measurements revealed poor stability of the DosT protein. Instability of ferricyanide-oxidized DosT and its tendency to precipitate also prevent an accurate assessment of its affinity for NO. Despite problems of stability with DosT, the efficiency of the reaction of oxy-DosT with NO in aerobic conditions could be documented using the slow NO donor DEA-NONOate (). Specifically, as a first equiv of NO is produced, the conversion of oxy-DosT to the Fe(III) state is confirmed by the decrease in Soret absorption at 413 nm and the loss of well-resolved α/β bands a 578 and 543 nm in favor of the less distinctive absorption features of the oxidized protein (). Subsequent production of NO beyond 1 equiv results in an increased Soret absorption at 419 nm (), an observation consistent with partial conversion of the Fe(III) state to Fe(III)-NO despite the competing reaction of molecular oxygen with NO. The amount of Fe(III)-NO formed in these experiments suggest an upper limit of 1 μM for the Kd in Fe(III) DosT.
Figure 7 Room temperature time course of the reaction of oxy-DosT with 1.33 equiv of DEA-NONOate at pH 7.4 (DEA-NONOate generates 1.5 equiv NO with a t1/2 ~ 16 min). Panel A shows the first 21 minutes of the reaction while panel B corresponds to the subsequent (more ...)