Catalytic mechanism and protein engineering of copper-containing
nitrite reductase
Wijma, Hein Jakob
Citation
Wijma, H. J. (2006, February 9). Catalytic mechanism and protein engineering of
copper-containing nitrite reductase. Retrieved from https://hdl.handle.net/1887/4302
Version:
Corrected Publisher’s Version
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Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
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Chapter
9
Concl
usi
ons
and
Future
Prospects
9.1 Catalysis by NiR
At physiological pH, the equilibrium constant strongly favours the nitrite
The equilibrium constant (Keq = [pAZUox][NO][pAZUred]-1[NO2-]-1) for the
conversion of nitrite to nitric oxide with pseudoazurin as the electron donor is determined by the midpointpotentials of the physiologicalelectron donor and the nitrite.In chapter 3,it is shown by calculation,and by experiment,thatthe midpointpotentialof the NO2-/NO(aq)
couple is 189 mV versus NHE in agreement with the commonly cited value of §350 mV (the latter value is for the equilibrium with gaseous nitric oxide at1 atmosphere (56,137)). Since at pH 7, the midpoint potentials of physiological electron donors are higher (≅ 260 mV versus NHE, chapter 3, Table 2), the equilibrium favours the substrates. Importantly, the midpoint potential of nitrite changes by -118 mV for each increase of the pH by one unit, which would shift the equilibrium constant by a factor of 100 per pH unit if the physiological electron donor had a constant midpoint potential. Although the midpoint potentialof pseudoazurin does decrease somewhatwith increasing pH,there is stilla very strong effect of pH on the equilibrium constant. This may be physiologically relevant as denitrification is fastestbetween pH 7 and 8 (158).
NiR catalyses bidirectionally, with a bias for nitrite reduction
In chapter 3 we show that also the reverse reaction is catalysed; around pH 7 the forward reaction is 6 times faster than the reverse reaction (chapter 3). An enzyme may catalyse the arrival at equilibrium faster in the forward than in the backward direction or vice versa (catalytic bias).In chapter 3 itis shown thatatpH 6.2 the equilibrium constantis 1 at which pH the kcat(forward)/kcat(reverse) > 30. Only above pH 7.5 is the reverse
Chapter 9
150
Nitrite reductase shows significant deviations from Michaelis-Menten kinetics
In chapter 4 of this thesis, it is shown that NiR does not follow Henry-Michaelis-Menten kinetics. Instead, the dependence on nitrite concentration resembles substrate inhibition below pH 6.5. A consequence of these deviations from Henry-Michaelis-Menten kinetics is that the pH at which catalytic activity is maximal depends on the nitrite concentration. The inhibiting effect of high nitrite concentration is due to the binding of a single nitrite to the oxidized type-2 site, which slows down the rate of electron transfer to the type-2 site. If the nitrite binds after reduction of the type-2 site, then the overall rate can be faster. Above pH 6.5, there is substrate activation, since the rate of type-1 to type-2 electron transfer without nitrite bound becomes slower due to the deprotonation of the Cu-bound H2O to OH-.
Nitrite can bind productively to both the oxidized and to the reduced type-2 site
As discussed in the introduction and in chapter 4, two different ordered mechanism for nitrite binding and electron transfer have been advanced in the literature, either nitrite binding first then reduction of the type-2 site or reduction first with nitrite binding afterwards. Here we find that the enzyme kinetics agree with a random sequential mechanism. A strong support for the model is that the KMB, which in the random sequential
mechanism is approximately equal to the Kd of the oxidized type-2 site for nitrite, has
indeed the same logarithmic dependence on pH. Furthermore, a random-sequential mechanism is in agreement with other data in the literature.
The reduced-Inactive State of the Type-2 Site is in slow equilibrium with the Reduced-Active State
In this thesis it is shown that the reduced type-2 site may occur in an active and in an inactive conformation (chapter 5). Earlier it was noticed that the reduced type-2 site does not bind nitrite (54). This observation underscores that metallo redox-centres may form inactive conformations depending on their redox-state. For example, in cd1 nitrite
reductase, the oxidized active site ends up mostly in an inactive conformation (255), and in NiFe-hydrogenase different inactive states are known (174). W hen a reduced-inactive conformation is made feasible for the type-1 site by engineering (chapter 6), then the inactivation is far faster than the inactivation of the type-2 site. W hile engineering metallo enzymes, it will be an important challenge to prevent such inactive conformations.
Future Steps
A logical continuation of the research described in this thesis would be to study the steady-state and pre-steady state kinetics of pseudoazurin with nitrite reductase. The onset for such work is provided by chapters 2-5. The optimized expression system for pseudoazurin provides good yields, sufficient to enable extensive experimentation. It may be possible to obtain valuable mechanistic information from the mutual dependence of the steady-state affinity and rate constants for pseudoazurin and nitrite on the other substrate its concentration. Subsequently, the proteins could be studied by stopped-flow (single-turnover/burst-kinetics) and rapid-quench EPR. A burst in product formation was observed upon mixing nitrite and NiR with reduced pseudoazurin (results not shown). With the dead-time of the presently available instrument (1 ms) the catalytic rates were too fast to obtain useful results (even at 0 °C). A cell with a shorter dead-time (0.2 ms) is now commercially available. One way to get more information out of the electrochemistry of immobilised NiR would be to replace the type-1 site by a CuA site. Functional CuA sites were already
engineered instead of type-1 sites several times (238, 256-258) and in NiR a CuA-like site
can even appear without protein engineering (84). CuA sites have a lower reorganization
energy giving faster electron transfer, which would help to supply electrons to the type-2 site faster, thereby enabling a more detailed investigation of the NiR by protein film voltammetry.
9.2 Engineering of External Ligands and Allosteric Effectors in Type-1 Copper Sites
In NiR an exogenous imidazole ligand can replace the C-terminal histidine in the oxidized state
Chapter 6 shows that in NiR H145A, the loss of the histidine ligand can be compensated for by the binding of an imidazole ligand when the type-1 copper is in the oxidized state. Upon reduction, imidazole dissociation is faster than electron transfer to the type-2 site. The dissociation raises the midpoint potential of the type-1 site. The results for NiR confirm results earlier obtained for azurin H117G (189, 197). The second order rate of reduction of NiR H145A-imidazole is almost the same as that of the wt (Table 4). In azurin H117G-imidazole the rate of reduction is 10-fold slower than the reduction of the wt azurin (55). This shows that also without a covalent bond the electron transfer can be fast.
Chapter 9
152
Furthermore, with an allosteric effector the type-1 site is fully functional (identical midpoint potential and reorganization energy as the wt NiR).
A methionine lowers the reorganization energy by 0.3 eV
Chapter 8 shows that in a type-1 site the methionine ligand lowers the reorganization energy by 0.3 eV. A lower reorganization energy due to the axial ligands of a type-1 site was indeed predicted by computational methods (259). The lower reorganization energy allows for faster electron transfer, which may have been an evolutionary pressure explaining the prevalence of a methionine as the axial ligand amongst type-1 Cu sites.
A method to measure ǻȜ of protein variants is now available
In chapter 8, a protocol is described to measure ǻȜ, the change of reorganization energy of a mutant relative to the wt protein. This can form a useful tool in the study of electron transfer proteins. Other methods consist of measuring the electron self exchange rates by NMR, which is difficult to apply to large (> 25 kDa) proteins, or by applying pulse-radiolysis, for which a second redox centre in the same protein is needed.
Future steps
