Deficiency of energy supply is a major complication contributing to the syndrome of heart failure (HF). As the concurrent activity profile of mitochondrial bioenergetic enzymes has not been studied collectively in human HF, our aim was to examine the mitochondrial enzyme defects in left ventricular myocardium obtained from explanted end-stage failing hearts. Compared to non-failing donor hearts, activity rates of complexes I and IV and the Krebs cycle enzymes isocitrate dehydrogenase, malate dehydrogenase and aconitase were lower in HF, as determined spectrophotometrically. However, activity rates of complexes II, III and citrate synthase did not differ significantly between the two groups. Protein expression, determined by western blotting, did not differ between the groups, implying post-translational perturbation. In the face of diminished total glutathione and coenzyme Q₁₀ levels, oxidative modification was explored as an underlying cause of enzyme dysfunction. Of the three oxidative modifications measured, protein carbonylation was significantly increased by 31% in HF (p<0.01; n=18), while levels of 4-hydroxynonenal and protein nitration though elevated, did not differ. Isolation of complexes I, IV and F₁F_oATP synthase by immunocapture revealed that proteins containing iron-sulphur or heme redox centres were targets of oxidative modification. Energy deficiency in end-stage failing human left ventricle involves impaired activity of key electron transport chain and Krebs cycle enzymes, without altered expression of protein levels. Augmented oxidative modification of crucial enzyme subunit structures implicates dysfunction due to diminished capacity for management of mitochondrial reactive oxygen species, thus contributing further to reduced bioenergetics in human HF.