Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers.
To understand the molecular mechanism of oxidation-induced inhibition of muscle contractility, we have studied the effects of hydrogen peroxide on permeabilized rabbit psoas muscle fibers, focusing on changes in myosin purified from these fibers. Oxidation by 5 mM peroxide decreased fiber contractility (isometric force and shortening velocity) without significant changes in the enzymatic activity of myofibrils and isolated myosin. The inhibitory effects were reversed by treating fibers with dithiothreitol. Oxidation by 50 mM peroxide had a more pronounced and irreversible inhibitory effect on fiber contractility and also affected enzymatic activity of myofibrils, myosin, and actomyosin. Peroxide treatment also affected regulation of contractility, resulting in fiber activation in the absence of calcium. Electron paramagnetic resonance of spin-labeled myosin in muscle fibers showed that oxidation increased the fraction of myosin heads in the strong-binding structural state under relaxing conditions (low calcium) but had no effect under activating conditions (high calcium). This change in the distribution of structural states of myosin provides a plausible explanation for the observed changes in both contractile and regulatory functions. Mass spectroscopy analysis showed that 50 mM but not 5 mM peroxide induced oxidative modifications in both isoforms of the essential light chains and in the heavy chain of myosin subfragment 1 by targeting multiple methionine residues. We conclude that 1) inhibition of muscle fiber contractility via oxidation of myosin occurs at high but not low concentrations of peroxide and 2) the inhibitory effects of oxidation suggest a critical and previously unknown role of methionines in myosin function.
Prochniewicz
Ewa
E
Department of Biochemistry, University of Minnesota, Jackson Hall 6-155, 321 Church St., Minneapolis, MN 55455, USA. ewa@ddt.biochem.umn.edu
Lowe
Dawn A
DA
Spakowicz
Daniel J
DJ
Higgins
LeeAnn
L
O'Conor
Kate
K
Thompson
LaDora V
LV
Ferrington
Deborah A
DA
Thomas
David D
DD
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D007537:Isometric Contraction / Q000187:drug effects / Q000502:physiology; D013058:Mass Spectrometry; D008715:Methionine / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D040901:Proteomics / Q000379:methods; D011817:Rabbits
D000199:Actins; D016877:Oxidants; D008715:Methionine; D006861:Hydrogen Peroxide; D000251:Adenosine Triphosphatases; D018995:Myosin Heavy Chains; D009218:Myosins