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Complex formation between the Escherichia coli [NiFe]-hydrogenase nickel maturation factors

  • Mozhgan Khorasani-Motlagh
  • Meissam Noroozifar
  • Kagan Kerman
  • Deborah B. ZambleEmail author


The biosynthesis of the dinuclear metal cluster at the active sites of the [NiFe]-hydrogenase enzymes is a multi-step process executed by a suite of accessory proteins. Nickel insertion during maturation of Escherichia coli [NiFe]-hydrogenase 3 is achieved by the metallochaperones HypA, SlyD and the GTPase HypB, but how these proteins cooperate to ensure nickel delivery is not known. In this study, the complexes formed between the individual purified proteins were examined by using several methods. Size exclusion chromatography (SEC) indicated that SlyD and HypB interact primarily in a 1:1 complex. The affinity of HypB-SlyD was measured by using surface plasmon resonance, which revealed a KD of 24 ± 10 nM in the absence of nucleotide and an interaction several fold tighter in the presence of GDP. A ternary complex between all three proteins was not detected, and instead SlyD blocked the interaction of HypA with HypB in competitive binding experiments. Furthermore, cross-linking experiments suggest a weak interaction between HypA and SlyD, which is not detectable by SEC. Electrochemical analysis confirmed each of the pairwise interactions and that the relative affinities of these complexes are on the order of HypB-SlyD > HypB-HypA > HypA-SlyD. These results indicate a hierarchy of interactions, as opposed to a single multiprotein complex, and provide insight into the nickel delivery process during hydrogenase enzyme maturation.


Hydrogenase maturation Nickel accessory proteins Metallochaperone interactions Nickel delivery 





Cyclic voltammetry


3,3′-Dithiobis(sulfosuccinimidyl propionate

E. coli

Escherichia coli


Electrochemical impedance spectroscopy


Guanosine diphosphate


Gold screen-printed electrode


Size exclusion chromatography



The authors would like to thank Michael Lacasse for constructive comments on the manuscript and Hashwin Ganesh for support with the SPR experiments. This work was supported in part by funding from the Canadian Institutes of Health Research (D.B.Z). K. K. acknowledges financial support from the Canada Research Chair Tier-2 program for “Bioelectrochemistry of Proteins”, the Ontario Ministry of Research and Innovation, the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Canada Foundation for Innovation.

Supplementary material

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Supplementary material 1 (PDF 1204 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of TorontoTorontoCanada
  2. 2.Department of BiochemistryUniversity of TorontoTorontoCanada
  3. 3.Department of Physical and Environmental SciencesUniversity of Toronto ScarboroughTorontoCanada
  4. 4.Department of ChemistryUniversity of Sistan and BaluchestanZahedanIran

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