Contribution of Backbone Dynamics to Entropy Changes Occurring on Oxidation of Cytochrome b₅. Can Redox Linked Changes in Hydrogen Bond Networks Modulate Reduction Potentials?

Abstract

Changes in backbone dynamics occurring upon oxidation of rat cytochrome b 5 have been examined through model free analyses of 15N-relaxation rates of both oxidation states of the protein. Based on the observed changes, an upper bound for the contribution of backbone dynamics to the entropy change associated with oxidation has been calculated. The magnitude of this backbone contribution, 70 ± 7 J/K·mol, is strikingly similar to the total entropy change associated with oxidation of the protein determined through an analysis of the temperature dependence of the reduction potential. Origins of the differences in dynamic behavior of the oxidized and reduced proteins can be attributed to redox linked changes in hydrogen bond strengths based on large-scale differences in amide proton exchange rates observed between the oxidation states. Based on these observations the magnitude and possible significance of entropic contributions to the electromotive force are discussed. Analysis of the 15N-relaxation rates included modeling of anisotropic diffusional behavior which was expected based on the distinct physical asymmetry of the protein. An axially symmetric diffusion tensor model was found to fit the rotational reorientational properties of the protein in both oxidation states. The contribution of paramagnetic relaxation to the 15N-relaxation rates of the oxidized protein was calculated based on a set of modified Solomon−Bloembergen equations. The determination of the electronic correlation time of the paramagnetic center was based on fits to the proton relaxation rate enhancements of protons in close proximity to the paramagnetic center. Analyses of the dynamic properties of the oxidized cytochrome b 5 were based on multiple field (i.e., 500 and 750 MHz) NMR measurements of 15N T 1 and T 2 relaxation times.

Fast structural dynamics in reduced and oxidized cytochrome c

“…The results presented here also suggest that redox‐dependent changes in residual protein entropy, expressed in the motions on the sub‐ns time scale,1, 13, 17, 36 are small in cytochrome c . This contrasts with cyt b 5 where significant changes in protein dynamics are apparent 31…”

The 1.2 Å structure of the human sulfite oxidase cytochromeb5domain

“…High crystallographic temperature factors can result from either dynamic disorder produced by intrinsic¯exibility of the atoms within the molecule or from static disorder in which residues adopt two or more alternate conformations. Residues 40±45 are within a region that exhibits considerable motion in other b 5 cytochromes and contributes signi®cantly to the entropic effect on the redox potential of the rat cytochrome b 5 domain (Dangi et al, 1998). Residues 41±43 of HSO b 5 are dynamically disordered; even at 1.2 A Ê resolution no discrete conformations can be resolved.…”

Cytochrome-P450–Cytochrome-b5 Interaction in a Membrane Environment Changes 15N Chemical Shift Anisotropy Tensors

“…The paramagnetic effect was found to be stronger due to an increase in the relaxation rate for amide protons and nitrogens in close proximity to the heme unit. 82 The mean CSA values of beta-sheet and loop-region residues are found to be smaller in the cytb 5 -cytP450 complex as compared to beta sheet ( σ ∥ – σ ⊥ =−166.0 ppm) and loop region residues ( σ ∥ – σ ⊥ =−161.1 ppm) in free cytb 5 . The decrease in CSA span in these regions of the cytb 5 -cytP450 complex could suggest a change in local motion in these regions of cytb 5 when it binds to cytP450 as compared to free cytb 5 .…”