References

4. Acknowledgement The authors thank the National Institutes of Health (Grant DK-37255 to W.E.N.) and the UK's BBSRC (to D.J.L.) for support.

SUBSTRATE REDUCTION AND CO SUSCEPTIBILITY OF THE ANIFV AND oc-Q191KMoFe PROTEINS OF AZOTOBACTER VINELANDII NITROGENASE

W.E. Newton, K. Vichitphan, K. Fisher

Dept of Biochemistry, The Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA

1. Introduction

Substrates and inhibitors interact at the FeMo-cofactor (FeMo-co) prosthetic group on the MoFe protein component of Azotobacter vinelandii. Homocitrate, the nijV-gene product, is an integral component of FeMo-co (Hoover et al. 1987) that ligates the Mo of FeMo-co, and hydrogen bonds with a-Glnl91. It may direct intra-MoFe-protein electron/proton transfer, substrate reduction and inhibitor binding, all of which may involve nearby amino-acid residues (Fisher et al. 2000). Insight into important acid-base groups within the MoFe protein arose from the effects of changing pH in the "3-buffers" system (Pham, Burgess 1993) on the H2-evolution rate +/-CO, using wild-type, a-Q191K and citrate-complemented (from the AnifV mutant) MoFe proteins. Unlike wild type, the last two suffer CO inhibition of H2 evolution.

2. Results and Discussion

We found:

(a) All three MoFe proteins produce bell-like curves, indicating two (at least) acid-base groups, one of which must be deprotonated (pKa ca. 6.0) and one protonated (pKa ca. 8.5) for nitrogenase activity. Above pH ca. 7, all three curves are misshapen, suggesting that more than one acid-base group contribute to the pKa of ca. 8.5.

(b) Because all six curves overlap below pH ca. 7, the group responsible for the pKa of ca. 6 is unaffected by both substitutions and CO and so is not likely to be a-Glnl91.

(c) The curves for both altered proteins are very similar (and different to wild type above pH 7.5) suggesting that a-Glnl 91-homocitrate is a component of the pKa at 8.5.

(d) The wild-type curve with CO more closely resembles those of the altered proteins without CO and suggests that: (i) substitutions in the a-Glnl 91-homocitrate system mimic the effect of added CO; (ii) CO "masks" a group's contribution to the pKa at 8.5; and (iii) CO binds close to a-Glnl91-homocitrate.

(e) The CO-induced shift in the pH-activity curve causes CO inhibition of H2 evolution for wild type (only above pH 7.5) and a-Q191K. No such shift occurs for ANifV, where inhibition occurs throughout the pH range, indicating a different mechanism of inhibition.

(f) With added CO, the a-Q191K MoFe protein produces a symmetrical bell-shaped curve, suggesting (with the above) that three groups contribute to the pKa of ca. 8.5.

3. References

Hoover TR et al. (1987) Nature 329, 855-857

Fisher K et al. (2000) Biochem. 39,10855-10865

Pham DN, Burgess BK (1993) Biochem. 32, 13725-13731

4. Acknowledgement

Support was from the NIH (#DK-37255).

NITROGENASE CATALYZED DINITROGEN REDUCTION - A NEW MECHANISTIC APPROACH

Dept of Chemistry, University of Iowa, Iowa City, IA 52242, USA

1. Introduction

The molecular mechanism of the enzymatic reduction of the inert gas N2 remains an open question, despite the immense amount of work that has been done in this area (Burgess, Lowe 1996; Rees, Howard 2000). We are conducting a mechanistic investigation of the dinitrogen (N2) reduction by the enzyme. Our goal is to develop a unique methodology that will enable the examination of the N2 reduction mechanism within the enzyme's complex kinetic cascade (Thorneley, Lowe 1983, 1996).

2. Procedure

A methodology is being established to measure competitive 15N kinetic isotope effects (KIEs). Competitive KIEs are effects on the second order rate constant V/K. These KIEs are only sensitive to kinetic steps from the free N2 binding to the first irreversible step (Cook 1991). Thus, these effects are not "masked" by the slow rate limiting steps. The enrichment of 15N in the remaining dinitrogen substrate is measured at various fractional conversions if) by isotope ratio mass spectrometry. The KIEs are calculated from: where Rt is the isotopic ratio at time t and Rq the isotopic ratio at t = 0 (Kohen, Klinman 1999; Melander, Saunders 1987). '5(r//;)_ ln0~/) Triple labeling 15N KIE experiments (15N2:15N14N:14N2) assist in the eluci- ln j _ fK)

dation of the intrinsic mechanism (Cook 1991). v^J

3. Analysis and Discussion

KIEs are a manifestation of changes in bond order along the reduction path. The experimental findings will be used to reevaluate various theoretical models suggested and will lead to the identification of model(s) consistent with the results. We hope to provide one of the first experimental tools to shed light on this fascinating chemical process. In the future, D20 and D2 effects on the 15N KIEs and studies with several mutants will enable examination of the reductive protonation of N2.

4. References

Burgess BK, Lowe DJ (1996) Chem. Rev. 96,2983-3011

Cook PF (ed.) (1991) Enzyme Mechanism From Isotope Effects, CRC Press, Boca Raton, FL Kohen A, Klinman JP (1999) Chem. Biol. 6, R191-198

Melander L, Saunders WH (1987) In Krieger RE (ed.) Reaction Rates of Isotopic Molecules, Malabar, FL

Rees DC, Howard JB (2000) Curr. Opin. Chem. Biol., 559-566 Thorneley RNF, Lowe DF (1983) Biochem J. 215, 393-403 Thorneley RNF, Lowe DF (1996) J. Biol. Inorg. Chem., 576-580

5. Acknowledgements

The wild-type Mo nitrogenase from Azotobacter vinelandii was a generous gift from B.K. Burgess. We thank R. Hoffmann for useful discussion regarding the theoretical models involved.

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