Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0-and 1.9-Å resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near ؊360 mV (assuming mitochondrial pH ϳ8.0 at 37°C). In other work (C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.

Comparative analysis of cytoplasmic organelles in a variety of tumors relative to normal tissues generally reveals a strong diminution in mitochondrial content and in oxidative phosphorylation capacity. However, little is known about what triggers these modifications and whether or not they are physiologically reversible. We hypothesized that energy substrate availability could play an important role in this phenomenon. The physiological effects of a change in substrate availability were examined on a human cancer cell line (HeLa), focusing specifically on its ability to use glycolysis versus oxidative phosphorylation, and the effect that energy substrate type has on mitochondrial composition, structure, and function. Changes in oxidative phosphorylation were measured in vivo by a variety of techniques, including the use of two novel ratiometric green fluorescent protein biosensors, the expression level of oxidative phosphorylation and some glycolytic enzymes were determined by Western blot, mitochondrial DNA content was measured by real-time PCR, and mitochondrial morphology was monitored by both confocal and electron microscopy. Our data show that the defective mitochondrial system described in cancer cells can be dramatically improved by solely changing substrate availability and that HeLa cells can adapt their mitochondrial network structurally and functionally to derive energy by glutaminolysis only. This could also provide an explanation for the enhancement of oxidative phosphorylation capacity observed after tumor regression or removal. Our work demonstrates that the pleomorphic, highly dynamic structure of the mitochondrion can be remodeled to accommodate a change in oxidative phosphorylation activity. We compared our finding on HeLa cells with those for nontransformed fibroblasts to help distinguish the regulatory pathways.

To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an extensive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human heart tissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individual protein components were separated by one-dimensional PAGE. Total in-gel processing and subsequent detection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct protein identifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. The coverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrial membrane was >90%. A significant proportion of identified proteins are involved in signaling, RNA, DNA, and protein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19% of the identified proteins have not been defined. This database of proteins provides a comprehensive resource for the discovery of novel mitochondrial functions and pathways.

Loss of mitochondrial complex I catalytic activity in the electron transport chain (ETC) is found in multiple tissues from individuals with sporadic Parkinson's disease (PD) and is a property of some PD model neurotoxins. Using special ETC subunit-specific and complex I immunocapture antibodies directed against the entire complex I macroassembly, we quantified ETC proteins and protein oxidation of complex I subunits in brain mitochondria from 10 PD and 12 age-matched control (CTL) samples. We measured nicotinamide adenine dinucleotide (NADH)-driven electron transfer rates through complex I and correlated these with complex I subunit oxidation levels and reductions of its 8 kDa subunit. PD brain complex I shows 11% increase in ND6, 34% decrease in its 8 kDa subunit and contains 47% more protein carbonyls localized to catalytic subunits coded for by mitochondrial and nuclear genomes We found no changes in levels of ETC proteins from complexes II-V. Oxidative damage patterns to PD complex I are reproduced by incubation of CTL brain mitochondria with NADH in the presence of rotenone but not by exogenous oxidant. NADH-driven electron transfer rates through complex I inversely correlate with complex I protein oxidation status and positively correlate with reduction in PD 8 kDa subunit. Reduced complex I function in PD brain mitochondria appears to arise from oxidation of its catalytic subunits from internal processes, not from external oxidative stress, and correlates with complex I misassembly. This complex I auto-oxidation may derive from abnormalities in mitochondrial or nuclear encoded subunits, complex I assembly factors, rotenone-like complex I toxins, or some combination.

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