Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002
Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539–552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 102–103 M−1 s−1 and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.
KeywordsTruncated hemoglobin 2/2 hemoglobin Heme posttranslational modification Hybrid b/c heme
Hemoglobin produced by Synechococcus sp. PCC 7002 or Synechocystis sp. PCC 6803
GlbN with covalently attached heme
GlbN with noncovalently attached heme
Heteronuclear single quantum coherence
This study was supported by National Science Foundation grant MCB-0349409. NMR facilities and resources at Johns Hopkins University were provided by the Biomolecular NMR Center. The authors thank Selena Rice for assistance with the optical measurements, Richard Himes, Ryan Peterson, and Kenneth Karlin for the use of their stopped-flow equipment, and Christopher Falzone for useful discussions and careful reading of the manuscript. Henry Nothnagel’s insight into heme chemistry was essential in the initial phases of the work. Figure 1a was prepared with Molscript .
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