XAS Study of Ni Enzyme - Story of CODH and ACS

W. Gu,1 H. X. Wang,1,2 J. Seravalli,3 S. W. Ragsdale,3 D. A. Grahame, 4 S. P. Cramer1,2

1University of California-Davis
2Lawrence Berkeley National Laboratory
3University of Nebraska - Lincoln
4Uniformed Services University of Health Sciences

Carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS or ACDS) are key enzymes to an autotrophic pathsway known as Wood/Ljungdahl pathway. CODH catalyzes the reversible oxidation of CO to CO2 and ACS is responsible for synthesis and cleavage of acetyl-CoA. The active site of the CODH (cluster C) and ACS (ACS) contains both Ni and Fe.

The crystal structures of CODH from C. hydrogenoformans (Ch-CODH) and R. rubrum (Rr-CODH) have been published. The two structures are similar. However, none of the structures have been predicted by previous Ni EXAFS studies of Rr-CODH. We thus studied the Ni EXAFS of Ch-CODH in two different states - as isolated and CO treated. There is a dramatic change in EXAFS while the as isolated sample is reduced by CO. The Ni EXAFS of as isolated can be simulated by 4 S at ~2.2 and no metal-metal interaction is observed. However for the CO treated sample, there is another peak at ~2.7 , which can be fit by at least 1 Ni-Fe, indicating that cluster reorganization occurs upon CO reduction. This also agrees with differences observed in the two crystallography data.

The beta subunit of methanogen ACDS contains cluster A. The isolated beta subunit (and the genetic recombinant) has the acetyl transfer activity. Element analysis shows that the Ni and Fe ratio of the subunit is 1 to 2. We studied recombinant beta subunit at both Fe and Ni K-edges under different redox conditions (as isolated and Ti(III) reduced). Fe EXAFS clearly shows that Fe is organized in a typical 4Fe-4S cluster. Ni EXAFS found that Ni is outside of the Fe-S cubane, and bound by 3S and 1N. Long distance interaction in Ni EXAFS is also observed, which could be interpreted by metal-metal interactions. Two possible structures for cluster A are proposed and discussed.

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