Characterization Of Nitrogenase y Protein Variants Generated By Sitedirected Mutagenesis

Department of Biochemistry, UW-Madison, WI, USA

Dinitrogenase is a heterotetrameric ((X2P2) enzyme that contains the iron-molybdenum cofactor (FeMo-co) at its active site. Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase, with a subunit composition 0^272, that can be activated in vitro by the addition of FeMo-co. The y subunit is able to interact with both FeMo-co and apodinitrogenase, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme.

The non-nif gene encoding the y protein has been recently cloned, sequenced, and found to encode a NifY-like protein that belongs to the NifY/NifX/VnfX family of iron and molybdenum (or vanadium) cluster-binding proteins (Rubio et al., manuscript in preparation). Comparison of their amino acid sequences pointed to the only conserved Cys (Cys166 in the y sequence) as a good candidate for cluster binding and, therefore, we have generated variants of the y protein with Ala or Ser in place of Cys166. Purified preparations of wild-type and mutant variants of y were used to compare their binding properties to FeMo-co and to apodinitrogenase. Results presented in this work are consistent with a role for Cys166 in stabilization of the y-FeMo-co complex.

ACETYLENE REDUCTION WITH AZOTOBACTER VINELANDII Mo-NITROGENASE: ROLE OF GLUTAMINE-191 IN a-SUBUNIT OF MoFe PROTEIN

K. Vichitphan, W.E. Newton

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

1. Introduction

The FeMo-cofactor (FeMo-co) is one of two types of prosthetic group found in the larger of the two nitrogenase component proteins, called the MoFe protein, and it is strongly implicated as the substrate binding and reduction site. The glutamine-191 residue in the a-subunit of the MoFe protein was targeted for substitution because it is located between the P cluster (the second type of prosthetic group in the MoFe protein) and the FeMo-co. Moreover, its side chain is involved in a hydrogen-bond network from one of the terminal carboxylate of the homocitrate component of FeMo-co through to the backbone NH of aGly-61, which is adjacent to the P cluster-ligating residue, aCys-62. The effect of substitution at the position a-191 on the properties of the FeMo-co could be mediated through hydrogen bonding and the homocitrate-Mo linkage. Substitution with lysine in this position, to give the aLys-191 altered MoFe protein, produces an unusual phenotype,

1.e. no nitrogen fixation, CO-sensitive H2 evolution, low C2H2 binding affinity, and C2H6 formation from C2H2 reduction (Scott et al. 1992; Fisher et al. 2000).

2. Results and Discussion

A variety of altered MoFe proteins, namely the aSer-191, aHis-191, aGlu-191, and aArg-191 altered MoFe proteins, have been purified to homogeneity and have the following results compared to wild type MoFe protein. All four altered MoFe proteins have decreased catalytic activity but charged amino-acid side chains decrease catalytic activity more (37% and 18% of wild type under Ar alone for aGlu-191 and aArg-191 altered MoFe protein, respectively). Based on a pH profile study, the decrease in catalytic activity could be due to a shift in the pKa of deprotonated and/or protonated group(s), resulting in changing pH for maximum activity. In the presence of 10% C2H2/90% Ar, the four altered MoFe proteins use less of the electron flux for C2H2 reduction (21-46%>) than does wild type (89%). The decreased rate of electron distribution to C2H2 reduction correlates with a decrease in C2H2 binding affinity (higher Km). Moreover, the aSer-191 altered MoFe protein exhibits a biphasic response for C2H2 reduction at pH 8.0 with two Km values (0.007 and 0.800 atm of C2H2 for C2H4 production), which suggest at least two C2H2 binding sites. The aHis-191, aGlu-191, and aArg-191 altered MoFe proteins produce ethane (C2H6) from C2H2 due to both their higher Km of C2H2 reduction and the pKa shift. These results suggest that the glutamine at the position a-191 has an effect on both the C2H2 binding site(s) and the pKa of responsible protonated/deprotonated group(s) during nitrogenase catalysis.

3. References

Scott DJ et al. (1992) J. Biol. Chem. 267, 20002-10 Fisher K et al. (2000) Biochem. 39, 29070-79

4. Acknowledgement

We thank Dr K. Fisher and the NIH (Grant DK-37255 to W.E.N).

AZOTOBACTOR VINELANDIINITROGENASE CONTAINING ALTERED MoFe PROTEINS WITH SUBSTITUTIONS AT a-278SER: INTERACTIONS AMONG SUBSTRATES AND INHIBITORS

Dept of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA

1. Introduction

FeMo-cofactor is one of two prosthetic groups bound within the MoFe protein of nitrogenase. The FeMo-cofactor's polypeptide environment appears to be intimately involved in the delicate control of the MoFe protein's interactions with its substrates and inhibitors (Fisher et ah 2000). In this work, the a-subunit 278-Serine residue of the MoFe protein was targeted. Altered MoFe proteins of Azotobactor vinelandii Mo-nitrogenase, the a-278Thr, a-278Cys, a-278Ala and a-278Leu MoFe proteins, were used to study interactions among H+, C2H2, CO and N2.

2. Results and Discussion

All strains except the a-278Leu mutant are Nif. We determined the Km of C2H2 reduction for the altered MoFe proteins. The a-278Ala and a- 278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a-278Thr and the a-278Leu MoFe proteins both have a Km ten times higher than the wild type for C2H2 reduction and, unlike wild type, both produce C2H6 These results suggest that the C2H2-binding site is affected by substitution at the a-278Ser position. Like the wild type, N2 is also a competitive inhibitor for the reduction of C2H2 by the a-278Thr, a-278Cys and a-278Ala MoFe proteins, but the Kj for N2 inhibition is higher than that with the wild type MoFe protein.

When reducing C2H2, the a-278Ala and a- 278Cys MoFe proteins respond to CO similarly to the wild type, whereas the a-278Thr MoFe protein is much more sensitive to CO. Under a nonsaturating concentration of CO, the a-278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between two C2H4-evolving sites. This phenomenon was previously observed with the a-277Hls MoFe protein, which has the a-277Arg substituted by histidine (Shen et ah 1997). These data suggest that the MoFe protein has two C2H2-binding sites, one of which appears to be located near the a-277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo-cofactor.

3. References

Shen et ah (1997) Biochem. 36, 4884-4894 Fisher K et ah (2000) Biochem. 39, 2970-2979

4. Acknowledgement

Supported by the NIH (DK-37255).

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