King, Clinton R

							                                King, Clinton R.
  The Effect of Salt Concentrations on A. vinelandii Nitrogenase
                   Faculty Mentor: Gary D. Watt, Chemistry and Biochemistry

We are investigating the kinetic effect of salt concentration on the reaction
  N2 + (6+2n)H+ + (6+2n)e- + 2(6+2n)ATP 2NH3 + nH2 + 2(6+2n)Pi + 2(6+2n)ADP                  (1)
catalyzed by nitrogenase where the spontaneous decomposition of S2O42- is the electron source.
Under high partial pressures of N2, n has a limiting value of one, but in the absence of N2,
nitrogenase simply catalyzes the reduction of protons to H2.

We observed that the concentrations of various salts (including buffers) affect the activity of the
nitrogenase system differently (Figure 1). The experiments that produced figure 1 were
performed under a nitrogen atmosphere. The difference in H2 production in the presence of
NH4Cl and NaCl suggest that the two salts interact differently with the protein. It also suggests
that the buffering system may make a difference. Indeed, that difference has been observed. A
protein with an activity of 1903 nmol H2 evolved/(min*mg protein) with 25mM TES (N-
tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffer was observed to have an activity
of 2410 with 30mM phosphate buffer.

In order to confirm these effects it is desirable to measure the NH3 production under these
conditions. Historical methods of measuring NH3 (NH4+ at this pH) have involved
microdistillation of the ammonia and a color producing reaction. These have proved unreliable.
We attempted to implement a method described by Dilworth et al,1 but this too proved
unsuccessful after many trials with standards. The measurement of small concentrations of
ammonium in the presence of large concentrations of sodium is a problem addressed by
manufacturers of ion chromatographic systems. Richens et al2 recently published a method of
quantifying ammonium in a large background by ion exchange chromatography. This research
was performed in Dr. Lamb’s laboratory in our own department. They have agreed to
collaborate with us and measure the ammonium in our samples. Figure 2 shows our expected
results.

An examination of Figure 2 shows that we wish to measure, in 1mL assays, µM changes of NH4+
in the presence 0–.5 M NH4Cl. We propose to accomplish this with the use of 15N2 as the
nitrogen source for (1). Because naturally occurring nitrogen is over 99% 14N, we will be able to
distinguish between the 14NH4+ initially present and any 15NH4+ that the reaction might produce.
Upon quenching, we will make the solution basic and release NH3 into the sealed atmosphere.
We will withdraw some gas from the headspace and inject it into a mass spectrometer, which has
the sensitivity to allow us to determine the amount of 15NH3 produced by nitrogenase in the
presence of NH4Cl.

Based on the Thorneley-Lowe3 kinetic theory of nitrogenase, the slow step of (1) is the
dissociation of the two nitrogenase proteins. We believe that the general salt effect accelerates
this step by decreasing the energy of dissociation. We believe that the specific effect of
NH4+/NH3 is to associate with the active site of nitrogenase and prevent N2 reduction.4
                                                                                  Salt Effect on H2 activity

                                                              8000
                      2/(min*mg
   specific activity (nmol H




                                                              7000

                                                              6000
                                    protein))




                                                              5000
                                                                                                                            NaCl
                                                              4000
                                                                                                                            NH4Cl
                                                              3000

                                                              2000

                                                              1000

                                                                0
                                                                     0   100      200        300        400     500   600
                                                                                   Salt Concentration (mM)



Figure 1 – Assays were run for ten minutes in sealed vials under a nitrogen atmosphere. Protein concentration in the
NaCl and NH4Cl experiments was 1.02 µM. An ATP regenerating system, excess Na2S2O4, and 25mM TES as a pH
buffer, at pH=7.4, was present.

                                                                           Theoretical Salt Effect on NH3 activity

                                                              4500
                                  evolved/(min*mg protein))
   specific activity (nmol NH




                                                              4000
                        3




                                                              3500

                                                              3000

                                                              2500                                                          NaCl
                                                              2000                                                          NH4Cl

                                                              1500

                                                              1000

                                                              500

                                                                0
                                                                     0   100      200        300        400     500   600
                                                                                   salt concentration (mM)



Figure 2 – The theoretical salt effect on NH3 production that we expect to verify.

References
                (1) Dilworth, M. J.; Eldridge, M. E.; and Eady, R. R. Correction for Creatine Interference
                    with the Direct Indophenol Measurement of NH3 in Steady-State Nitrogenase Assays.
                    Anal. Biochem. 1992, 207, 6–10.
                (2) Richens, D. A.; Simpson, D.; Peterson, S.; McGinn, A.; and Lamb, J. D. Use of mobile
                    phase 18-crown-6 to improve peak resolution between mono- and divalent metal and
                    amine cations in ion chromatography. J Chrom. A. 2003, 1016, 155–164.
                (3) Lowe, D. J.; and Thorneley, R. N. F. The Mechanism of Klebsiella pneumoniae
                    Nitrogenase Action. Biochem. J. 1984, 224, 877–907.
                (4) The author wishes to acknowledge interpretive help of Phil E. Wilson, PhD candidate.