The structure of zinc (II)-substituted cytochrome c (Zn-Cytc), recently resolved by NMR, suggested that Zn is in an unusual coordination with six ligands (Anni et al., Biochemistry 1995, 34, 5744-5753). Furthermore, fluorescence line narrowing spectroscopy has shown that Zn becomes penta-coordinated upon protein unfolding. An hexa-coordinated state of Zn in Cytc is unique since there are no examples of any hexa-coordinated Zn-proteins, and there are no hexa-coordinated Zn-porphyrins in solution. Moreover, the particular coordination of Zn with five nitrogens and one sulfur ligand in Cytc is surprising, because even among the Zn organic compounds that are hexa-coordinated (27% of the 428 crystal structures) none is of the type (N) 5-Zn-(S) 1. Taken together these findings point to a tight protein modulation of the binding preference and strength of Zn to its ligands in Cytc. The Zn-edge extended X-Ray absorption fine structure H(EXAFS) studies of Zn-Cytc were undertaken in order to define the Hcentral metal environment, specifically the distances of the Zn metal Hto its ligands and their nature. Both native and guanidine-denatured HZn-Cytc at neutral pH were measured, alongside with the corresponding HFe(III)-Cytc references. Sets of experimental EXAFS data were fit to Htheoretical curves (FEFF 6.01) using a novel refinement procedure were Hobtained. This data analysis establishes the coordination of the Zn Hsite in Cytc: an hexa-coordinated Zn in the native protein Henvironment (Zn-Np=2.052A, Zn-Met80 (SD) =2.34A, Zn-His(NE)=2.371A) Hand a mixture of penta-coordinated Zn states in the denatured sample H(Zn-Np=2.10A, Zn-His(NE) =2.10-2.20A). Fe-cyt-c data showed close Hagreement with crystallographic data. Analysis of denatured Hcytochrome-c is continuing.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001633-17
Application #
6205757
Study Section
Project Start
1999-09-01
Project End
2000-08-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
17
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
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