QUT ePrints

Heterologous overexpression and purification of cytochrome c' from Rhodobacter capsulatus and a mutant (K42E) in the dimerization region. Mutation does not alter oligomerization but impacts on heme iron spin state and nitric oxide binding properties

Huston, Wilhelmina M., Andrew, Colin R., Sevid, Amy E., McKay, Alison L., Leech, Andrew P., Butler, Clive S., & Moir, James W. (2006) Heterologous overexpression and purification of cytochrome c' from Rhodobacter capsulatus and a mutant (K42E) in the dimerization region. Mutation does not alter oligomerization but impacts on heme iron spin state and nitric oxide binding properties. Biochemistry, 45(14), pp. 4388-4395.

View at publisher

Abstract

Rhodobacter capsulatus cytochrome c¢ (RCCP) has been overexpressed in Escherichia coli, and its spectroscopic and ligand-binding properties have been investigated. It is concluded that the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteins are both monomeric in solution, contrary to the homodimeric structure of the crystalline state. Surprisingly, the K42E mutation produces a number of changes at the heme center (nearly 20 Å distant), including perturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex from a six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectra indicate that ferrous K42E and wild-type RCCP both have relatively high Fe-His stretching frequencies, suggesting that the more favored five-coordinate heme-nitrosyl formation in K42E is not caused by a weaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with its modified ferric spin state, shows that structural changes originating at the dimer interface can affect the properties of the heme center, raising the exciting possibility that intermolecular encounters at the protein surface might modulate the reactivity of cytochrome c¢ in vivo.

Impact and interest:

5 citations in Scopus
Search Google Scholar™
5 citations in Web of Science®

Citation countsare sourced monthly from Scopus and Web of Science® citation databases.

These databases contain citations from different subsets of available publications and different time periods and thus the citation count from each is usually different. Some works are not in either database and no count is displayed. Scopus includes citations from articles published in 1996 onwards, and Web of Science® generally from 1980 onwards.

Citations counts from the Google Scholar™ indexing service can be viewed at the linked Google Scholar™ search.

ID Code: 7158
Item Type: Journal Article
Additional Information: This article is freely available from the American Chemical Society website 12 months after the publication date. See links to publisher website in this record.
Additional URLs:
Keywords: Rhodobacter capsulatus, cytochrome c', heme spin state
DOI: 10.1021/bi052605j
ISSN: 0006-2960
Subjects: Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > MICROBIOLOGY (060500) > Microbiology not elsewhere classified (060599)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > MICROBIOLOGY (060500) > Microbial Genetics (060503)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > MICROBIOLOGY (060500) > Bacteriology (060501)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > BIOCHEMISTRY AND CELL BIOLOGY (060100) > Biochemistry and Cell Biology not elsewhere classified (060199)
Divisions: Past > QUT Faculties & Divisions > Faculty of Science and Technology
Current > Institutes > Institute of Health and Biomedical Innovation
Copyright Owner: Copyright 2006 American Chemical Society
Deposited On: 27 Apr 2007
Last Modified: 29 Feb 2012 23:28

Export: EndNote | Dublin Core | BibTeX

Repository Staff Only: item control page