Modulation of ligand–heme reactivity by binding pocket residues demonstrated in cytochrome c' over the femtosecond–second temporal range

Authors

  • Henry J. Russell,

    1. Faculty of Life Sciences, Manchester Institute of Biotechnology and Photon Science Institute, The University of Manchester, UK
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  • Samantha J. O. Hardman,

    1. Faculty of Life Sciences, Manchester Institute of Biotechnology and Photon Science Institute, The University of Manchester, UK
    2. Central Laser Facility, Research Complex at Harwell, Didcot, UK
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  • Derren J. Heyes,

    1. Faculty of Life Sciences, Manchester Institute of Biotechnology and Photon Science Institute, The University of Manchester, UK
    2. Central Laser Facility, Research Complex at Harwell, Didcot, UK
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  • Michael A. Hough,

    1. School of Biological Sciences, University of Essex, UK
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  • Gregory M. Greetham,

    1. Central Laser Facility, Research Complex at Harwell, Didcot, UK
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  • Michael Towrie,

    1. Central Laser Facility, Research Complex at Harwell, Didcot, UK
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  • Sam Hay,

    1. Faculty of Life Sciences, Manchester Institute of Biotechnology and Photon Science Institute, The University of Manchester, UK
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  • Nigel S. Scrutton

    Corresponding author
    1. Faculty of Life Sciences, Manchester Institute of Biotechnology and Photon Science Institute, The University of Manchester, UK
    2. Central Laser Facility, Research Complex at Harwell, Didcot, UK
    • Correcpondence

      N. S. Scrutton, Faculty of Life Sciences, Manchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, UK

      Fax: +44 161 3068918

      Tel: +44 161 3065152

      E-mail: nigel.scrutton@manchester.ac.uk

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  • [The copyright line was changed on 2 May 2014 after original online publication]

Abstract

The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide (NO) and carbon monoxide (CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue (R124) and a distal leucine residue (L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond–second temporal range in the UV-vis spectrum, and the femtosecond–nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124A variant. In the R124A variant, vibrational and electronic changes extend continuously across all time scales (from fs–s), in contrast to wild-type cytochrome c' and the L16A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124A variant of cytochrome c' that is distinct from that in wild-type cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range.

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