Alternate layer-by-layer polyion adsorption onto gold electrodes coated with chemisorbed mercaptopropanesulfonic acid gave stable, electroactive multilayer films containing the proteins myoglobin and cytochrome P450(cam). Direct, reversible, electron exchange between gold electrodes and proteins involved heme Fe-III/Fe-II redox couples. With oxygen in solution, electrons were also transferred to the Fe-II-O-2 complexes of these proteins, a key step for oxidative enzyme catalysis. Film assembly for Mb was done by sequential adsorption with poly(styrenesulfonate) (PSS), DNA, or poly(dimethyl diallyl) ammonium chloride (PDDA). Cyt P450(cam) was assembled with layers of PSS or PDDA. Quartz crystal microbalance and voltammetric studies on the same films allowed quantitation of electroactive and nonelectroactive protein. At pH 5.5, the first protein monolayer in all films was fully electroactive. A second monolayer added 30-40% redox activity, but additional protein layers did not communicate with the electrode. Using various film construction strategies, Mb monolayers were also placed at distances from the electrodes of 0.5; 1.8, and 3.8 nm. Full electroactivity was found at 0.5 nm, and about 70-80% electroactivity at 1.8 and 3.8 nm. Results suggest the possibility of enhanced electron transport by partial intermixing of protein and nonprotein layers. Polyion films containing Mb and cyt P450(cam) were active for enzyme-like catalysis of styrene epoxidation in aerobic solutions.